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Volume 40, No. 4 July/August 2019
ISSN 0143-2044
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Protection by
glycerol, sucrose and trehalose for acellular human dermis against gamma irradiation damage at -78.5°C
Wendell Q. Sun
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200-208
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Prediction of
cryoprotectant permeation into articular cartilage: a model suitable for several cryoprotective agents
Xiaoyi Yu and Guangming Chen
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209-218
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Rosmarinus officinalis essential oil preloaded in β-cyclodextrin: effect on ram spermatozoa motility, membrane integrity and oxidative status during 4°c storage
A. Benberkane, A. Khellouf, K. Benhenia, S. Fatmi
and M. Iguer-ouada
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219--225
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The viability of serval (Leptailurus serval)
and Pallas cat (Felis manul) oocytes after cryopreservation using the Rapid-I method
Agnieszka Nowak, Joanna Kochan, Sylwia Prochowska,
Agnieszka Partyka, Wiesława Młodawska,
Wojciech Witarski, Józef Skotnicki, Teresa Grega,
Marcin Pałys and Wojciech Niżański
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226-230
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In vitro and
in vivo efficiency of extenders added to thawed stallion semen
Felipe Augusto Boudoux Martins Sales,
José Carlos Ferreira-Silva, Joane Isis Travassos Vieira,
Maiana Silva Chaves, Eduardo Luiz Cavalcanti Caldas,
José Pompeu Santos Filho, Vicente José Figuêiredo Freitas
and Marcos Antonio Lemos Oliveira
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231-236
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Cryopreservation
of in vitro-grown shoot tips of the medicinal species Cleome spinosa (Cleomaceae) applying vitrification-based techniques
Anna Flávia Rodrigues Mortani Vilardo,
Thaís Fortunato Mendonça, Florent Engelmann,
Lívia Silva Cordeiro, Norma Albarello
and Claudia Simões-Gurgel
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237-246
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Vapour
pressure above the glassy trehalose solution and glass relaxation
Shaozhi Zhang, Xiao Niu, Gengsheng Huang,
Guangming Chen and Xiangguo Xu
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247-256
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CryoLetters 40 (4), 200-208 (2019)
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PROTECTION BY GLYCEROL, SUCROSE AND TREHALOSE FOR ACELLULAR HUMAN DERMIS AGAINST GAMMA IRRADIATION DAMAGE AT -78.5°C
Wendell Q. Sun
Institute of Biomedical Technology, School of Medical Instrument and Food Engineering,
University of Shanghai for Science and Technology, Shanghai, China.
For correspondence: wendell.q.sun@gmail.com
Abstract
BACKGROUND: Glycerol, sucrose and trehalose are used as protectants for
membrane, protein, cell and tissue preservation. The undercooled state (glassy or rubbery) of their solutions may also offer protection for protein, cells and tissues against
radiation damage upon sterilization. OBJECTIVE: The present study aimed to examine the protective effects of glycerol, sucrose and trehalose on cryopreserved acellular
human dermis against gamma irradiation damage. MATERIALS AND METHODS: Acellular human dermis was cryopreserved at -80°C in glycerol, sucrose and trehalose
solutions or their combinations with a base citrate-phosphate buffer (pH 6.0). Cryopreserved acellular dermis was then subjected to 13 kGy gamma irradiation at
-78.5aC, and radiation damage was assessed by histological evaluation. RESULTS: Freeze and thaw alone do not alter the structure of acellular dermis, but gamma irradiation at -78.5°
C results in significant structural changes in acellular dermis, including the formation of large holes, the damage of collagen fibers and the loss of
overall dermis tissue histology. The incorporation of glycerol, sucrose and trehalose into cryopreservation solutions reduces gamma irradiation-induced tissue structural damage
considerably. When used alone, trehalose (0.5 M) provided better protection against gamma irradiation damage than did sucrose (0.5 M) and glycerol (1.0 M). When used in
combination, the glycerol/trehalose combination provides the best tissue protection. Significant donor-to-donor variation exists in tissue damage after gamma irradiation. For
donor dermis that is less sensitive to gamma irradiation damage, glycerol, sucrose or trehalose alone is able to provide good protection. However, for more sensitive donor
dermis, only the glycerol/trehalose combination is able to provide sufficient tissue protection. CONCLUSION: Glycerol, sucrose and trehalose protects cryopreserved
acellular human dermis against gamma irradiation damage. Cryopreservation solutions can be optimized to permit tissues for gamma sterilization to increase the safety human tissue implants.
Keywords: cryopreservation, gamma irradiation, acellular dermis, glycerol, sucrose, trehalose.
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CryoLetters 40 (4), 209-218 (2019)
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PREDICTION OF CRYOPROTECTANT PERMEATION INTO ARTICULAR CARTILAGE: A MODEL SUITABLE FOR SEVERAL CRYOPROTECTIVE AGENTS
Xiaoyi Yu* and Guangming Chen
Ningbo Institute of Technology, Zhejiang University, Ningbo, China
*Corresponding author email: yuxiaoyi@nit.zju.edu.cn
Abstract
BACKGROUND: Understanding the spatiotemporal distribution characteristics of
cryoprotectant concentration in the cartilage tissue during loading is essential for the rational design of the loading procedure and the successful cryopreservation of articular cartilage (AC).
OBJECTIVE: To construct a diffusion mass transfer model to describe the permeation of cryoprotectant into AC. MODEL CONSTRUCTION: The model was
based on the binary diffusion thermodynamic model to correlate the effective diffusion coefficient with the infinite dilution diffusion coefficient. The activity coefficient was
estimated using the UNIFAC model. The concentration dependence of the diffusion coefficient was expressed using the Vignes formula, and the infinite dilution diffusion
coefficient was calculated using the Siddiqi-Lucas correlation. RESULTS: For dimethyl sulfoxide (Me2SO), glycerol (GLY), ethylene glycol (EG) and propylene glycol (PG), the
coefficient of determination and mean relative error of the average cryoprotectant concentration in the cartilage tissue calculated by the model and experimental values
reported in the literature are 0.959–0.998 and 0.98–15.19%, respectively, for Me2SO; 0.990–0.995 and 13.56–19.19% for GLY; 0.969–0.988 and 8.89–22.09% for EG; and
0.971–0.992 and 5.35–23.76% for PG. CONCLUSION: The developed model could be used for these four widely used cryoprotectants to directly predict the spatiotemporal
distribution of cryoprotectant concentration in the cartilage tissue during loading.
Keywords: mass transfer, diffusion, articular cartilage, cryoprotectant, UNIFAC, cryopreservation
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CryoLetters 40 (4), 219-225 (2019)
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Rosmarinus officinalis ESSENTIAL OIL PRELOADED IN Β-CYCLODEXTRIN:
EFFECT ON RAM SPERMATOZOA MOTILITY, MEMBRANE INTEGRITY AND OXIDATIVE STATUS DURING 4°C STORAGE
A. Benberkane 1*, A. Khellouf 1, K. Benhenia 2,
S. Fatmi 3 and M. Iguer-ouada 1
1Associated Laboratory in Marine Ecosystems and Aquaculture, Department of
Biological Sciences of the Environment, Faculty of Nature and Life Sciences, Abderrahmane Mira University, Route de Targua Ouzemmour, 06000 Bejaia Algeria.
2National Center for Biotechnology Research (CRBt), Ali Mendjli Nouvelle Ville UV 03
BP E73, Constantine, Algeria.
3Pharmaceutical Laboratory, Department of Engineering Processes, Abderrahmane Mira University, Route de Targua Ouzemmour, 06000 Bejaia, Algeria.
*Corresponding author: ben_amine92@live.fr
Abstract
BACKGROUND: Rosmarinus officinalis essential oil (Rom) has been reported recently
to be of interest for use in sperm cryopreservation. However, related to its lipophilic characteristics, encapsulation in cyclodextrin could enhance Rom positive effects by
increasing its solubility in sperm extenders. OBJECTIVE: To compare the effect of Rom preloaded in hydroxypropyl-β-cyclodextrin (Rom-cd) to Rom alone (Rom) on ram sperm conserved at 4°C.
MATERIALS AND METHODS: Ram epididymal sperm was collected from six testes. Each collected sperm was split into four equal aliquots. The control
aliquot was diluted with Tris extender (Tris + citric acid + fructose + penicillin), two aliquots were treated with Rom at 0.5 μl ml-1 and 1 μl ml-1 respectively, and one aliquot
treated with Rom-cd at 1 μl ml-1. All sperm aliquots were analyzed for motility after 0, 2, 4, 24 and 48 h of storage at 4°C using a Computer Aided Semen Analysis (CASA).
Membrane integrity and oxidative stress status were measured after 48 h of storage. RESULTS: The results indicated that motility parameters were best preserved in the
extender containing Rom-cd compared to the groups treated by Rom without cyclodextrin. Rom alone resulted to higher sperm motility than the control group. Lower
oxidative stress and more cell membrane protection were observed in Rom treated samples, especially when using Rom-cd. CONCLUSION: The ability of Rom to protect
ram sperm against cryopreservation damages was improved after encapsulation in hydroxypropyl-β-cyclodextrin (HPβCD).
Keywords: ram sperm, cryopreservation, Rosmarinus officinalis, essential oil, β-cyclodextrin, solubility.
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CryoLetters 40 (4), 226-230 (2019)
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THE VIABILITY OF SERVAL (Leptailurus serval) AND PALLAS CAT (Felis manul)
OOCYTES AFTER CRYOPRESERVATION USING THE RAPID-I METHOD.
Agnieszka Nowak1, Joanna Kochan1 *, Sylwia Prochowska2,
Agnieszka Partyka2, Wiesława Młodawska1, Wojciech Witarski3,
Józef Skotnicki4, Teresa Grega4, Marcin Pałys4
and Wojciech Niżański2
1 University of Agriculture in Krakow, Faculty of Animal Sciences, Institute of Veterinary
Sciences, al. Mickiewicza 21, 30-120 Krakow, Poland.
2 Wrocław University of Environmental and Life Sciences, Faculty of Veterinary
Medicine, Department of Reproduction and Clinic of Farm Animals, Poland.
3 National Research Institute of Animal Production, Department of Genomics and Animal Molecular Biology, Poland.
4 Foundation Municipal Park and the Zoological Garden in Cracow, Poland.
* Corresponding author: joanna.kochan@urk.edu.pl
Abstract
BACKGROUND: Vitrification by Rapid-I method could be essential for felid rescue programs to protect wild felid in the future. OBJECTIVE:
This study was aimed at adapting the Rapid I method and evaluating the viability of serval and Pallas cat oocytes compared to oocytes of the domestic cat. MATERIALS AND METHODS: Oocytes after
collection and in vitro maturation were vitrified using Cryotech medium (Cryotech, Japan) and a Rapid-I device (Vitrolife, Sweden). To evaluate viability, oocytes after warming
were stained with fluorescein diacetate and ethidium bromide. RESULTS: Survival rate in the control group (domestic cat) was 75 %. In the experimental group, 70% (serval)
and 60% (pallas cat) viable oocytes were found. CONCLUSION: The Rapid-I method can be applied successfully for the vitrification of wild felid oocytes.
Keywords: wild felids, oocyte, Rapid-I, vitrification, bio-bank
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CryoLetters 40 (4), 231-236 (2019)
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IN VITRO AND IN VIVO EFFICIENCY OF EXTENDERS ADDED TO THAWED STALLION SEMEN
Felipe Augusto Boudoux Martins Sales1, José Carlos Ferreira-Silva1,
Joane Isis Travassos Vieira1, Maiana Silva Chaves1,
Eduardo Luiz Cavalcanti Caldas2, José Pompeu Santos Filho1,
Vicente José Figuêiredo Freitas3 and Marcos Antonio Lemos Oliveira1*
1 Laboratório de Biotécnicas Reprodutivas, Universidade Federal Rural de Pernambuco, Recife-PE, Brasil.
2 Universidade Federal de Sergipe, São Cristóvão-SE, Brasil.
3 Universidade Estadual do Ceará, Fortaleza-CE, Brasil.
* Corresponding author: maloufrpe@gmail.com
Abstract
BACKGROUND: Addition of extenders to thawed semen could improve fertility. OBJECTIVE:
To determine the efficiency of extenders to increase viability of thawed semen, measured by sperm parameters in vitro and pregnancy rates after artificial insemination (AI). MATERIALS AND METHODS:
Sperm motility and acrosin activity were measured during a thermoresistance test (TRT). RESULTS: Progressive motility
decreased (P<0.05) after 30 min in thawing semen treated with saline solution (SS) and only after 60 min with Tyrode's solution (TS) or freezing diluent (FD). The total motility
decreased (P<0.05) after 60 min in thawed semen treated with SS, and after 90 min in thawed semen containing TS or FD. The acrosin activity decreased (P<0.05) after 60
min during the TRT, but there was no difference among treatments throughout the TRT. The pregnancy rates were similar among thawed-semen supplemented with SS, TS or FD. CONCLUSION:
The extenders neither improve sperm parameters nor enhance AI results.
Keywords: equine, cryopreservation, post-diluent, motility, acrosin, fertility.
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CryoLetters 40 (4), 237-246 (2019)
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CRYOPRESERVATION OF IN VITRO-GROWN SHOOT TIPS OF THE MEDICINAL SPECIES Cleome spinosa (Cleomaceae) APPLYING VITRIFICATION-BASED
TECHNIQUES
Anna Flávia Rodrigues Mortani Vilardo1*, Thaís Fortunato Mendonça1,
Florent Engelmann2, Lívia Silva Cordeiro1, Norma Albarello1
and Claudia Simões-Gurgel1
1 Laboratório de Biotecnologia de Plantas (LABPLAN) - Núcleo de Biotecnologia
Vegetal (NBV) - Universidade do Estado do Rio de Janeiro (UERJ). Rua São Francisco Xavier, 524, PHLC, sala 509, Maracanã, CEP 20550-013 - Rio de Janeiro, RJ, Brazil.
2 Institut de Recherche pour le Développement (IRD), 08 BP 841 Cotonou, Benin.
*Corresponding author: anna.flavia.vilardo@gmail.com
Abstract
BACKGROUND: Few cryopreservation studies have been reported with the genus Cleome. Due to the use of C. spinosa in traditional medicine and its valuable
pharmacological potential, the long-term conservation of the species will allow the safe maintenance of its germplasm. OBJECTIVE: This study compares two
vitrification-based techniques on the cryopreservation of shoot tips of C. spinosa. MATERIALS AND METHODS: The effect of sucrose preculture and different vitrification
solutions was evaluated using vitrification and V Cryo-plate techniques. The supplementation of recovery medium with BAP was also assessed. RESULTS: The V
Cryo-plate proved to be the most efficient technique. Treatment of shoot tips with PVS2 at 0°C resulted in a higher regeneration response after cryopreservation when compared
to treatment with PVS2 and PVS3 at 25°C. The highest survival (83.3%) and recovery (76.6%) were achieved for shoot tips exposed to PVS2 for 90 min at 0°C and recovered
on MS medium supplemented with 0.5 mg L−1 BAP for 2 weeks. CONCLUSION: Plants regenerated from cryopreserved shoot tips maintained their in vitro multiplication
capacity and showed a normal phenotypic aspect, demonstrating the efficiency of the cryopreservation protocol.
Keywords: BAP, long-term conservation, PVS, shoot tips, vitrification, V Cryo-plate.
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CryoLetters 40 (4), 247-256 (2019)
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VAPOUR PRESSURE ABOVE THE GLASSY TREHALOSE SOLUTION AND GLASS RELAXATION
Shaozhi Zhang a,b, Xiao Niu a, Gengsheng Huang a,
Guangming Chen a, Xiangguo Xu a*
a Key Laboratory of Refrigeration and Cryogenic Technology of Zhejiang Province,
Institute of Refrigeration and Cryogenics, Zhejiang University, Hangzhou, P.R. China.
b National Quality Inspection Center of Refrigeration Equipment (Henan), Minquan, P.R. China.
*Corresponding author: zjuxgxu@zju.edu.cn
Abstract
BACKGROUND: Data are scarce on thermophysical properties of the glassy trehalose solution at low temperatures. OBJECTIVE:
Water vapor pressure above the glassy trehalose solution and the relaxation behavior were studied at temperatures from -57°C to -40°C and at concentrations from 71% to 78% (w/w).
MATERIALS AND METHODS: Glassy trehalose solutions were prepared by quenching in liquid nitrogen. Vapour pressure was measured using the static method. RESULTS: Vapour pressure above the
glassy trehalose solution was slightly lower than above the glassy sucrose solution. The relaxation of the glassy state can be described by the stretched exponential
Kohlrausch-Williams-Watts (KWW) function. The characteristic times of water relaxation (τp) were compared with those of enthalpy relaxation (τe). When the
difference (ΔT) between the glass transition temperature (Tg) and ageing temperature
was relatively small, i.e. ΔT ≤ 10 K, τp is close to τe. If ΔT ≥ 15 K, τe will be much greater than τp. CONCLUSION:
The difference of water vapor pressure above the trehalose glassy solution and sucrose glassy solution could lead to significant distinction between their drying kinetics.
Keywords: glass ageing, glass relaxation, trehalose solution, vapour pressure, water activity.
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