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Volume 36, No. 6 November/December 2015
ISSN 0143-2044
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Effects
cryopreservation of ionotropic glutamatergic receptor mechanisms in vitro
A.A. Mokrushin
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353-362
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Design of a
simple slow cooling device for cryopreservation of small biological samples
Leonardo Juan de Paz, María Celeste Robert,
Daniel Adolfo Graf, Edgardo Elvio Guibert and
Joaquin Valentin Rodriguez
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363-371
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Bacteria
in cryopreserved sperm mass of the white shrimp Penaeus vannamei
Karina Morales-Ueno, Carmen Guadalupe Paniagua-Chávez,
Rebeca Vasquez-Yeomans, Jorge A. Cáceres-Martinez and
Miguel A. Del Rio-Portilla
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372-378
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Ultrastructural
response of embryonic axes of Fortunella polyandra to dehydration and cryopreservation
Omar M. Al Zoubi and Normah M.N.
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379-391
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A simple and
efficient vitrification method for in-straw dilution and direct transfer of bovine embryos
Youwen Zhang, Xiangwei Fu, Long Chen, Chuntao Feng,
Jianghua Bi, Xianhong Mo, Keren Cheng, Rina Zhang,
Shujing Li and Shien Zhu
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392-398
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Germplasm
banking and its role in the development of the fish genetic improvement programme in Brazil
D. P. Streit Jr., D. C. Fornari, J. A. Povh, L. C. Godoy,
F de Mello, C. A. L. Oliveira, E. Kawakami and R. P. Ribeiro
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399-404
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Effect
of semen extender on protein concentration in each fraction of cryopreserved human semen
Vickram A S, Kamini Rao, Ramesh Pathy M,
Chanchal Thomas, Parameswari R and Sridharan T B
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405-412
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Determination
of heat transfer coefficients for french plastic semen straw suspended in static nitrogen vapor over liquid nitrogen
M.V. Santos, M. Sansinena, J. Chirife and N. Zaritzky
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413-423
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Authors Index
Keyword Index
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424-426
427-429
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CryoLetters 36 (6), 353-362 (2015)
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EFFECTS CRYOPRESERVATION OF IONOTROPIC GLUTAMATERGIC RECEPTOR MECHANISMS IN VITRO
A.A. Mokrushin
I. P. Pavlov Institute of Physiology, Russian Academy of Science, 199034, Nab.
Makarova, 6, Saint-Petersburg, Russia
Corresponding author e-mail: mok@inbox.ru
Abstract
BACKGROUND:
We assayed hypothesis of cryopreservation of small volumes of nervous tissue (olfactory cortex slices). OBJECTIVE: Effect of freezing (-10°C) and rewarming to +37°
C studied on glutamatergic mechanisms. MATERIALS AND METHODS: Viability slices determined from the change of the amplitudes of
glutamatergic synaptic transmission: action potential of lateral olfactory tract (AP LOT), α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPAR) and N-methyl-D-aspartate (NMDAR) receptor-dependent mechanisms. Effects of two rates
freezing/rewarming: slow (0.1°C/min) and rapid (9.0°C/min) were studied. RESULTS: Activity AP LOT and AMPARs restored at slow rate freezing/rewarming, and at rapid
rate, these indicators were increased. Activity NMDARs decreased at slow rate both at slow and at rapid rates after freezing/rewarming were blocked. At rewarming water
swelling content ofin slices was maximal at rapid rate of freezing/rewarming. CONCLUSION: The protocol of cryopreservation with slow rate (0.1°C/min)
freezing/rewarming and a depth of freezing -10°C is optimal for the cryopreservation of small volumes of nervous tissue.
Keywords:
brain slices, freezing/rewarming, AMPARs, NMDARs
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CryoLetters 36 (6), 363-371 (2015)
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DESIGN OF A SIMPLE SLOW COOLING DEVICE FOR CRYOPRESERVATION OF SMALL BIOLOGICAL SAMPLES
Leonardo Juan de Paz1,2, María Celeste Robert1, Daniel Adolfo Graf1,2, Edgardo
Elvio Guibert1 and Joaquin Valentin Rodriguez1*
1 Centro Binacional (Argentina -Italia) de Investigaciones en Criobiología Clínica y
Aplicada (CAIC), Universidad Nacional de Rosario, Avda. Arijón 28 bis (S2011BXN) Rosario, Argentina.
2Servicio de Electrónica y Óptica, Facultad de Ciencias Bioquímicas y
Farmacéuticas, Universidad Nacional de Rosario, Suipacha 531 (S2002LRK) Rosario, Argentina.
*Corresponding author email: jrodrig@fbioyf.unr.edu.ar
Abstract
BACKGROUND:
Slow cooling is a cryopreservation methodology where samples are cooled to its storage temperature at controlled cooling rates. OBJECTIVE: Design,
construction and evaluation of a simple and low cost device for slow cooling of small biological samples. MATERIALS AND METHODS: The device was constructed based
on Pye's freezer idea. A Dewar flask filled with liquid nitrogen was used as heat sink and a methanol bath containing the sample was cooled at constant rates using copper bars as heat conductor.
RESULTS: Sample temperature may be lowered at controlled cooling rate (ranging from 0.4ºC/min to 6.0ºC/min) down to ~-60ºC, where it could be
conserved at lower temperatures. An example involving the cryopreservation of Neuro-2A cell line showed a marked influence of cooling rate over post preservation
cell viability with optimal values between 2.6 and 4.6°C/min. CONCLUSION: The cooling device proved to be a valuable alternative to more expensive systems allowing
the assessment of different cooling rates to evaluate the optimal condition for cryopreservation of such samples.
Keywords:
slow cooling device, cooling rate, cryopreservation, neural cells
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CryoLetters 36 (6), 372-378 (2015)
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BACTERIA IN CRYOPRESERVED SPERM MASS OF THE WHITE SHRIMP PENAEUS VANNAMEI
Karina Morales-Ueno, Carmen Guadalupe Paniagua-Chávez1*,
Rebeca Vasquez-Yeomans2, Jorge A. Cáceres-Martinez1 and
Miguel A. del Rio-Portilla1
1Aquaculture Department, Centro de Investigación Científica y de Educación Superior
de Ensenada, B.C. (CICESE) Carretera Ensenada-Tijuana No. 3918 Zona Playitas, Ensenada, B. C. Mexico CP 22840.
2Insitituto de Sanidad Acuícola, Calle de la marina S/N esquina caracoles,
Fraccionamiento Playa Ensenada, Ensenada, B. C. Mexico CP 22880.
*Corresponding author email: cpaniagu@cicese.mx
Abstract
BACKGROUND:
Cryopreservation and global trading of P. vannamei sperm will become a potential and important biotechnological tool. Nevertheless, information of
the possible transfer of bacteria in cryopreserved shrimp sperm has not been registered yet. OBJECTIVE: The objective of this work was to determine the type of
bacteria that could be cryopreserved together with white shrimp sperm masses. MATERIALS AND METHODS: Sixteen sperm masses were cryopreserved in 10%
DMSO and 0.5 M trehalose and sixteen fresh sperm masses were used for bacterial analysis. Bacterial colonies were isolated and selected for sequencing. RESULTS:
Strains were seawater borne and facultative aerobic bacteria from the genera Bacillus, Micrococcus, Paracoccus, Ruegeria and Staphylococcus. Most of them have been
related with benefits to its host. None were pathogenic for P. vannamei. CONCLUSION: Cryopreservation implies preserving pathogenic or beneficial bacteria
together with the sample. Therefore, it is possible to enhance cryopreserved samples or disperse pathogenic bacteria, which needs to be prevented.
Keywords:
shrimp, sperm mass, risk, bacteria, cryopreservation
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CryoLetters 36 (6), 379-391 (2015)
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ULTRASTRUCTURAL RESPONSE OF EMBRYONIC AXES OF FORTUNELLA
POLYANDRA TO DEHYDRATION AND CRYOPRESERVATION
Omar M. Al Zoubi1* and Normah M.N.2
1School of Biosciences and Biotechnology, Faculty of Science and Technology,
Universiti Kebangsaan Malaysia, 43600 UKM, Bangi, Selangor, Malaysia.
2Institute of Systems Biology, Universiti Kebangsaan Malaysia, 43600 UKM, Bangi, Selangor, Malaysia.
*Corresponding author email: omaralzoubi73@yahoo.com
Abstract
BACKGROUND:
To further understand the survival characteristics of desiccation-sensitive excised embryonic axes of Fortunella polyandra to desiccation
and cryopreservation it is necessary to study the impact of drying rates on both the ultrastructure and electrolyte leakage. OBJECTIVE: To examine the effects of two
different drying regimes (silica gel and ultra-rapid) on the survival, ultrastructure and membrane leakage characteristics of excised embryonic axes of F. polyandra before
and after cryopreservation. MATERIALS AND METHODS: The effects of the drying regimes on the survival, ultrastructure and membrane integrity of the excised embryonic axes of F. polyandra was determined. Survival was assessed in vitro, and
the integrity of membranes following drying was estimated by electrolyte leakage and observation under the transmission electron microscope (TEM). Survival and
ultrastructural changes were also observed after cryopreservation. RESULTS: Electrolyte leakage increased with decreasing water content of the embryonic axes,
indicative of substantial subcellular damage, after both ultra-rapid dehydration (to water contents <0.16 g H2O g-1 dw) and silica gel dehydration (to <0.28 g H2O g-1 dw water
content). Ultrastructurally, axes showed increasing cytoplasm and vacuole shrinkage and disruption of cell membranes with longer dehydration periods. Normal seedling
recovery of 50 to 47% for cryopreserved embryonic axes of F. polyandra was observed after ultra-rapid and silica gel drying respectively. Extreme cell injury was observed
after exposure to liquid nitrogen at high moisture content. Although cells of dehydrated axes encountered stress during cryopreservation, the main damage occurred during the dehydration step.
CONCLUSION: For surviving axes, the damage was less severe and the axes grew to become normal seedlings. Ultrastructural studies reveal the
damage of the cells at different rates of dehydration and during cryopreservation.
Keywords:
dehydration rate, electrolyte conductivity, survival, ultrastructure.
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CryoLetters 36 (6), 392-398 (2015)
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A SIMPLE AND EFFICIENT VITRIFICATION METHOD FOR IN-STRAW DILUTION AND DIRECT TRANSFER OF BOVINE EMBRYOS
Youwen Zhang1, Xiangwei Fu1, Long Chen2, Chuntao Feng2,
Jianghua Bi2, Xianhong Mo3, Keren Cheng1, Rina Zhang1,
Shujing Li2 and Shien Zhu1*
1Key Laboratory of Animal Genetics, Breeding and Reproduction, Ministry of
Agriculture and National Engineering Laboratory for Animal Breeding, College of Animal Science and Technology, China Agricultural University, Beijing 100193, P.R. China.
2Bingjing AnBo Embryo Biotech Center, Beijing 100107, P.R. China.
3College of Biological Sciences, China Agricultural University, Beijing 100193, P.R. China.
*Corresponding author email: zhushien@cau.edu.cn
Abstract
BACKGROUND:
An easy and user friendly protocol that produces consistent results will facilitate the commercial application of embryo vitrification technology in the field. OBJECTIVE:
This study was designed to develop a simple and efficient vitrification, in-straw dilution and direct transfer method for bovine embryos. METHODS: After being vitrified and in-straw thawed, in vivo-derived and in vitro-produced bovine embryos
were subjected to in vitro culture or embryo transplantation. RESULTS: There were no significant differences (Pâ¾0.05) in survival rates (100.0% vs. 93.9%) and expansion rates (93.8% vs. 87.5%) between in vivo-derived and in vitro-produced blastocysts after
vitrification and in-straw dilution. And there was also no significant difference (Pâ¾0.05) in conception rates (56.5% vs. 58.8%) after ET between cryopreserved and fresh in vivo-derived blastocysts. CONCLUSION: Vitrification using EG-based
vitrification solution and in-straw dilution with PBS-based diluent is a simple and efficient method for cryopreservation and direct transfer of bovine embryos.
Keywords:
Bovine, IVF, blastocyst, vitrification, in-straw dilution, embryo transfer
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CryoLetters 36 (6), 399-404 (2015)
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GERMPLASM BANKING AND ITS ROLE IN THE DEVELOPMENT OF THE FISH GENETIC IMPROVEMENT PROGRAMME IN BRAZIL
D. P. Streit Jr.1, D. C. Fornari2, J. A. Povh3, L. C. Godoy4,
F de Mello1*, C. A. L. Oliveira5, E. Kawakami6 and R. P. Ribeiro5
1Federal University of Rio Grande do Sul - UFRGS, Department of Animal Science,
Porto Alegre, Brazil.
2Genetic Fish Rise, Sorriso, Brazil.
3Federal University of Mato Grosso do Sul - UFMS, Faculty of Veterinary Medicine
and Animal Science, Campo Grande, Brazil.
4Aquaculture Graduate Program, Nilton Lins University/INPA, Manaus, Brazil.
5Maringá State University - UEM, Department of Animal Science, Maringá, Brazil.
6EMBRAPA-CNPAP, Corumbá, Brazil.
*Corresponding author email: Fernandade.mello@gmail.com
Abstract
BACKGROUND:
Over the last ten years, Brazilian fish farming has become more focused, resulting in the development of genetic improvement programmes (GIP) for two South American species Colossoma macropomum (tambaqui) and Pseudoplatystoma reticulatum (cachara).
OBJECTIVE: To describe the action plan used for setting up the GIP and to detail the germplasm bank composition. MATERIALS AND METHODS: Semen of both species was collected, frozen and
transported between locations in Brazil. To start the programme, full and half-sib families of both species were established from 120 males and 60 females. RESULTS:
New species-specific protocols for semen cryopreservation s were established of value to commercial application in fish farming. CONCLUSION: Germplasm banking has
enabled the exchange of biological material and reduced the overall GIP costs. Germplasm banking can be very important to the dissemination of the selected genetic
material of these species among fish farmers.
Keywords:
Semen cryopreservation, freezing solutions, Sperm, Tambaqui, Cachara
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CryoLetters 36 (6), 405-412 (2015)
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EFFECT OF SEMEN EXTENDER ON PROTEIN CONCENTRATION IN EACH FRACTION OF CRYOPRESERVED HUMAN SEMEN
Vickram A S, Kamini Rao, Ramesh Pathy M, Chanchal Thomas,
Parameswari R and Sridharan T B*
Department of Industrial Biotechnology, School of Biosciences and Technology, VIT
University, Vellore, India.
*Corresponding author email: tbsridharan@vit.ac.in
Abstract
BACKGROUND:
Semen extender is liquid diluents which are added to semen to preserve its fertilizing ability. The addition of extender to semen protects the sperm
against possible damage by toxic seminal plasma, as well as providing nutrients. AIM AND OBJECTIVE: The hypotheses of the study are to evaluate, if there is any
significant change in the protein concentration in each fractions of human semen before and after freezing with prepared semen extender. Then we are going to correlate
the semen parameters with thee fresh semen total protein concentration. RESULTS: After preserving with the extender, all the semen samples shows statistically significant (p<0.001) in case of protein concentration.
CONCLUSION: There is a significant different (p<0.001) in protein concentration in various fractions before and after freezing. Also the prepared semen extender can be used in Assisted
Reproductive centres (ART).
Key words:
Antioxidants, frozen, glycerol, protein, semen extender.
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CryoLetters 36 (6), 413-423 (2015)
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DETERMINATION OF HEAT TRANSFER COEFFICIENTS FOR FRENCH PLASTIC SEMEN STRAW SUSPENDED IN STATIC NITROGEN VAPOR OVER LIQUID NITROGEN
M.V. Santos 1,3*, M. Sansinena2,3, J. Chirife2 and N. Zaritzky1,2
1Depto. de Ingeniería Química, Facultad de Ingeniería, Universidad Nacional de La
Plata and Centro de Investigación y Desarrollo en Criotecnología de Alimentos (CONICET-UNLP) Calle 47 y 116, La Plata 1900, Argentina.
2Facultad de Ciencias Agrarias, Pontificia Universidad Católica Argentina, Cap. Gral.
Ramón Freire 183, CABA 1426, Argentina.
3Consejo Nacional de Investigaciones Científicas y Técnicas. Av. Rivadavia 1917, CABA
*Corresponding author email: mvsantosd@gmail.com
Abstract
BACKGROUND:
The use of mathematical models describing heat transfer during the freezing process is useful for the improvement of cryopreservation protocols. A
widespread practice for cryopreservation of spermatozoa of domestic animal species consists of suspending plastic straws in nitrogen vapor before plunging into liquid
nitrogen. Knowledge of surface heat transfer coefficient (h) is mandatory for computational modelling; however, h values for nitrogen vapor are not available.
OBJECTIVE: In the present study, surface heat transfer coefficients for plastic French straws immersed in nitrogen vapor over liquid nitrogen was determined; vertical and
horizontal positions were considered. MATERIALS AND METHODS: Heat transfer coefficients were determined from the measurement of time-temperature curves and
from numerical solution of heat transfer partial differential equation under transient conditions using finite elements. The h values experimentally obtained for horizontal
and vertically placed straws were compared to those calculated using correlations based on the Nusselt number for natural convection. RESULTS: For horizontal straws
the average obtained value was h=12.5 1.2 W/m2 K and in the case of vertical straws h=16 2.48 W/m2 K. The numerical simulation validated against experimental
measurements, combined with accurate h values provides a reliable tool for the prediction of freezing curves of semen-filled straws immersed in nitrogen vapor. CONCLUSION:
The present study contributes to the understanding of the cryopreservation techniques for sperm freezing based on engineering concepts, improving the cooling protocols and the manipulation of the straws.
Keywords:
plastic French straw, heat transfer coefficient, nitrogen vapor, mathematical model, cryopreservation, freezing
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