scholarly journals Two-Step Freezing Procedure for Cryopreservation of Rumen Ciliates, an Effective Tool for Creation of a Frozen Rumen Protozoa Bank

2003 ◽  
Vol 69 (7) ◽  
pp. 3826-3832 ◽  
Author(s):  
E. Nsabimana ◽  
S. Kišidayová ◽  
D. Macheboeuf ◽  
C. J. Newbold ◽  
J. P. Jouany
Keyword(s):  
Liquid Nitrogen ◽  
Ice Nucleation ◽  
Slow Freezing ◽  
Nucleation Temperature ◽  
Subzero Temperature ◽  
Equilibration Time ◽  
Rumen Protozoa ◽  
Ciliate Species ◽  
Freezing Procedure ◽  
Holding Temperature

ABSTRACT The present study aimed at the long-term storage of rumen protozoa as living cells in liquid nitrogen. The two-step or interrupted slow freezing procedure was used to cryopreserve six of the dominant species of rumen ciliates isolated from monofaunated animals, Dasytricha ruminantium, Entodinium caudatum, Epidinium ecaudatum caudatum, Eudiplodinium maggii, Isotricha prostoma, and Polyplastron multivesiculatum. We optimized the first step in the interrupted slow freezing procedure, from the extracellular ice nucleation temperature to the holding temperature, and studied the effects of the cooling rates on survival. In addition to the nature of the cryoprotectant (dimethyl sulfoxide), the equilibration temperature and equilibration time (25°C and 5 min, respectively), and the holding time at subzero temperature (45 min) recommended previously (S. Kišidayová, J. Microbiol. Methods 22:185-192, 1995), we found that a holding temperature of −30°C, a cooling rate from extracellular ice nucleation temperature to holding temperature of between 1.2°C/min and 2.5°C/min, depending on the ciliate, and rumen juice as the freezing and thawing medium markedly improved the survival rate. Survival rates determined after 2 weeks in liquid nitrogen were 100% for Isotricha, 98% for Dasytricha, 85% for Epidinium, 79% for Polyplastron, 63% for Eudiplodinium, and 60% for Entodinium. They were not significantly modified after a period of 1 year in liquid nitrogen. Four of the five ciliate species cryopreserved for 8 months in liquid nitrogen successfully colonized the rumen when inoculated into defaunated animals. These results have made it possible to set up a bank of cryopreserved rumen protozoa.

High ice nucleation temperature of zebrafish embryos: slow-freezing is not an option

Cryobiology ◽  
2004 ◽  
Vol 49 (2) ◽  
pp. 181-189 ◽  
Author(s):  
M. Hagedorn ◽  
A. Peterson ◽  
P. Mazur ◽  
F.W. Kleinhans
Keyword(s):  
Ice Nucleation ◽  
Slow Freezing ◽  
Nucleation Temperature ◽  
Zebrafish Embryos

Preservation of Morphological Integrity and Clot Retraction Activity of Human Platelets After Freezing

1964 ◽  
Vol 11 (01) ◽  
pp. 222-229 ◽  
Author(s):  
Isaac Djerassi ◽  
Albert Roy ◽  
Jorge Alvarado ◽  
Keyword(s):  
Dimethyl Sulfoxide ◽  
Liquid Nitrogen ◽  
Human Platelets ◽  
Slow Freezing ◽  
Critical Conditions ◽  
Plasma Medium ◽  
Clot Retraction ◽  
Controlled Freezing ◽  
Direct Immersion

SummaryHuman platelets frozen at −195° C (liquid nitrogen) retain their morphological integrity and ability to promote clot retraction when 5% dimethyl-sulfoxide and 5% dextrose are added to the suspending plasma medium. Slow freezing was more effective than direct immersion in the liquid nitrogen. Although similar results may be achieved with dimethylsulfoxide alone with rigidly controlled freezing rates, the addition of sugars may permit freezing under less critical conditions.Dimethylsulfoxyd und 5% Dextrose dem Plasmamilieu hinzugefügt werden. Das langsame Einfrieren ist effektiver als das direkte Eintauchen in flüssigen Stickstoff. Obschon ähnliche Resultate mit Dimethylsulfoxyd allein unter exakter Kontrolle der Einfrierungsgeschwindig-keit erreicht werden können, erlaubt die Zugabe von Dextrose ein Einfrieren unter weniger kritischen Bedingungen.

132 SUCCESSFUL EMBRYO TRANSFER OF IN VIVO-PRODUCED RED DEER (CERVUS ELAPHUS) EMBRYOS AFTER CRYOPRESERVATION BY SLOW FREEZING OR VITRIFICATION

2007 ◽  
Vol 19 (1) ◽  
pp. 183
Author(s):  
J. P. Soler ◽  
G. G. Kaiser ◽  
N. Mucci ◽  
L. B. Ferre ◽  
R. H. Alberio
Keyword(s):  
Liquid Nitrogen ◽  
Embryo Transfer ◽  
Cervus Elaphus ◽  
Red Deer ◽  
Slow Freezing ◽  
Alternative Methods ◽  
Embryo Cryopreservation ◽  
Chi Square ◽  
Cryopreserved Embryos

Multiple ovulation and embryo transfer (MOET) programs for red deer (Cervus elaphus) have been established commercially over the last decade, with embryo cryopreservation being a related practice necessary to enhance the use of valuable genetic information. The aim of this work was to establish alternative methods for red deer embryo cryopreservation by using slow freezing with ethylene glycol (SF–EG) and vitrification by open pulled straw (OPS) methods. After surgical flushing of 18 superstimulated donors, 54 transferable embryos were recovered; 28 were transferred fresh to synchronized recipients and the others were cryopreserved by SF–EG (n = 11) or OPS (n = 15), respectively thawed or warmed, and transferred to recipients. Fresh embryos were maintained in Dulbecco's PBS + 20% cow serum (holding medium, HM) until transfer (maximum 3 h after collection). SF–EG cryopreserved embryos were suspended in HM + 1.78 M EG + 0.1 M sucrose + 4 mg mL−1 BSA. After a 10-min equilibration, embryos were loaded individually into 0.25-mL plastic straws and placed into a −7°C methanol bath chamber. After seeding (5 min later), the straws were cooled from −7 to −35°C at a rate of 0.5°C min. Straws were plunged into and stored in liquid nitrogen. Thawing was performed by placing the straws in a 30°C water bath for 30 s; their contents were drained into HM until transfer. Embryos were vitrified using the OPS method with minor modifications. They were first incubated in HM + 1.78 M EG + 1.3 M DMSO for 3 min and then transferred for 25 s into a vitrification solution of HM + 3.56 M EG + 2.6 M DMSO + 0.5 M sucrose. Each embryo was loaded by touching a 1-µL drop with the straw, which was immediately submerged into and stored in liquid nitrogen. Warming was done by placing the narrow end of the straws into HM + 0.25 M sucrose for 5 min. Embryos were then transferred into HM + 0.15 M sucrose for 5 min and finally to HM until transfer. Both types of cryopreserved embryos were transferred a few hours after collection, immediately after thawing or warming. Before embryo transfer, the presence of corpus luteum (CL) of recipients was confirmed by laparoscopic examination. Each embryo was surgically transferred into the apical extreme of the uterine horn ipsilateral to the CL of one recipient. Pregnancy was determined by ultrasonography 41 days after embryo transfer. The pregnancy rate between groups was compared with the chi-square test (P < 0.05). No statistical differences were found between groups (Table 1). Our results show that both vitrification and slow freezing methods with EG are suitable to cryopreserve red deer embryos. Table 1. Pregnancy rates in recipient hinds after transfer of fresh, vitrified, or frozen red deer embryos

scholarly journals Characterization of aerosol particles at Cabo Verde close to sea level and at the cloud level – Part 2: Ice-nucleating particles in air, cloud and seawater

2020 ◽  
Vol 20 (3) ◽  
pp. 1451-1468 ◽  
Author(s):  
Xianda Gong ◽  
Heike Wex ◽  
Manuela van Pinxteren ◽  
Nadja Triesch ◽  
Khanneh Wadinga Fomba ◽  
...  
Keyword(s):  
Sea Level ◽  
Cloud Water ◽  
Ice Nucleation ◽  
Cape Verde ◽  
Nucleation Temperature ◽  
Sea Spray ◽  
Cloud Droplets ◽  
Cabo Verde ◽  
Active Particles ◽  
Sea Surface Microlayer

Abstract. Ice-nucleating particles (INPs) in the troposphere can form ice in clouds via heterogeneous ice nucleation. Yet, atmospheric number concentrations of INPs (NINP) are not well characterized, and, although there is some understanding of their sources, it is still unclear to what extend different sources contribute or if all sources are known. In this work, we examined properties of INPs at Cabo Verde (a.k.a. Cape Verde) from different environmental compartments: the oceanic sea surface microlayer (SML), underlying water (ULW), cloud water and the atmosphere close to both sea level and cloud level. Both enrichment and depletion of NINP in SML compared to ULW were observed. The enrichment factor (EF) varied from roughly 0.4 to 11, and there was no clear trend in EF with ice-nucleation temperature. NINP values in PM10 sampled at Cape Verde Atmospheric Observatory (CVAO) at any particular ice-nucleation temperature spanned around 1 order of magnitude below −15 ∘C, and about 2 orders of magnitude at warmer temperatures (>-12 ∘C). Among the 17 PM10 samples at CVAO, three PM10 filters showed elevated NINP at warm temperatures, e.g., above 0.01 L−1 at −10 ∘C. After heating samples at 95 ∘C for 1 h, the elevated NINP at the warm temperatures disappeared, indicating that these highly ice active INPs were most likely biological particles. INP number concentrations in PM1 were generally lower than those in PM10 at CVAO. About 83±22 %, 67±18 % and 77±14 % (median±standard deviation) of INPs had a diameter >1 µm at ice-nucleation temperatures of −12, −15 and −18 ∘C, respectively. PM1 at CVAO did not show such elevated NINP at warm temperatures. Consequently, the difference in NINP between PM1 and PM10 at CVAO suggests that biological ice-active particles were present in the supermicron size range. NINP in PM10 at CVAO was found to be similar to that on Monte Verde (MV, at 744 m a.s.l.) during noncloud events. During cloud events, most INPs on MV were activated to cloud droplets. When highly ice active particles were present in PM10 filters at CVAO, they were not observed in PM10 filters on MV but in cloud water samples instead. This is direct evidence that these INPs, which are likely biological, are activated to cloud droplets during cloud events. For the observed air masses, atmospheric NINP values in air fit well to the concentrations observed in cloud water. When comparing concentrations of both sea salt and INPs in both seawater and PM10 filters, it can be concluded that sea spray aerosol (SSA) only contributed a minor fraction to the atmospheric NINP. This latter conclusion still holds when accounting for an enrichment of organic carbon in supermicron particles during sea spray generation as reported in literature.

Cryopreservation of Human Nasal Ciliated Epithelium

1996 ◽  
Vol 10 (5) ◽  
pp. 291-298 ◽  
Author(s):  
Bin Yang ◽  
Thomas V. McCaffrey ◽  
Eugene B. Kern
Keyword(s):  
Liquid Nitrogen ◽  
Room Temperature ◽  
Storage Time ◽  
Beat Frequency ◽  
Ciliary Beat Frequency ◽  
Slow Freezing ◽  
Freezing And Thawing ◽  
Ciliary Function ◽  
Nasal Cilia ◽  
Fast Freezing

The purpose of this study was to compare the effects on human nasal cilia of various freezing and thawing methods in order to determine a reliable method of cryopreservation. Ten samples each were preserved by a slow freezing and a fast freezing method. All samples were stored in liquid nitrogen (–196°C) for 1 week. The frozen samples were thawed by one of two methods: 1) rapid thawing—37°C water bath for 3–4 minutes; and 2) slow thawing—room temperature for 15 minutes. Prefreeze and postthaw ciliary beat frequency (CBF) was measured. The slow freezing and fast thawing method (SFFT) resulted in the best viability. An additional 10 samples preserved using the SFFT method stored at –70°C to –90°C did not retain ciliary function. Thirty ciliated samples preserved by the SFFT method were examined after freezing in liquid nitrogen (–196°C) for 1 week, 2 weeks, and 1 month. There was no significant decrease in CBF after cryopreservation at –196°C for 1 week or 2 weeks (P> 0.05). As the storage time increased to 1 month, postthaw CBF decreased 7.25 ± 0.87% when compared to the prefreeze CBF (P < 0.05). We conclude that human nasal cilia preserved by SFFT at –196°C retain activity for up to 1 month.

scholarly journals Ice Nucleation, Freezing Injury, and Colonization of `Totem' Strawberry Flowers with Ice-nucleation-active (INA) Bacteria

1998 ◽  
Vol 123 (2) ◽  
pp. 234-238 ◽  
Author(s):  
Michele R. Warmund ◽  
James T. English
Keyword(s):  
Pseudomonas Syringae ◽  
Ice Nucleation ◽  
Deionized Water ◽  
Freezing Injury ◽  
Nucleation Temperature ◽  
Ice Formation ◽  
Fragaria Ananassa ◽  
Naturally Occurring ◽  
Ina Bacteria ◽  
Wetness Period

INA bacteria were isolated from primary flowers of `Totem' strawberry (Fragaria ×ananassa Duch.) plants that had been previously inoculated with strain Cit 7 of Pseudomonas syringae van Hall or noninoculated to determine their relationship to ice-nucleation temperature and floral injury. Mean ice-nucleation temperature of inoculated and noninoculated flowers was -2.2 and -2.8 °C, respectively. Primary flowers of noninoculated plants survived lower temperatures than those of inoculated plants. In another experiment, noninoculated plants were misted with sterile deionized water and incubated for 0, 12, 24, 36, or 48 hours at 25 °C day/10 °C night, and naturally occurring INA bacteria were isolated from primary flowers. INA bacterial densities increased exponentially with increasing incubation period. The critical wetness period for INA bacteria to establish a sufficient density to increase the likelihood of floral injury at -2.5 °C was 24 hours. Longer wetness periods resulted in higher INA bacterial densities but did not increase the floral mortality rate. Thermal analysis demonstrated that the ice nucleation temperature was associated with strawberry floral injury. Thus, low temperature survival of flowers was adversely affected by moisture for ≥24 h due to the presence of a sufficient density of INA bacteria to incite ice formation and floral injury.

Investigation of the Effect of Oscillating Magnetic Field on Fresh-Cut Pineapple and Agar Gel as a Model Food During Supercooling Preservation

2019 ◽  
Vol 62 (5) ◽  
pp. 1155-1161 ◽  
Author(s):  
Taiyoung Kang ◽  
Jae-Young Her ◽  
Raymond Hoptowit ◽  
Marisa M. Wall ◽  
Soojin Jun
Keyword(s):  
Magnetic Field ◽  
Ice Nucleation ◽  
Cellular Damage ◽  
Subzero Temperature ◽  
Crystal Formation ◽  
Agar Gel ◽  
Supercooled State ◽  
Model Food ◽  
Oscillating Magnetic Field ◽  
Fresh Cut

Abstract. In this study, the effect of an oscillating magnetic field (OMF) on the supercooling of fresh-cut pineapple and agar gel was investigated. The pineapple was preserved in a supercooled state at -7°C, and its supercooled state was maintained for 14 days with the presence of the OMF, whereas the untreated sample was spontaneously nucleated within 24 h. Agar gel was prepared as equivalent to the soluble solids content of pineapple and showed a comparable supercooling behavior when exposed to a similar OMF treatment. Examination of the microstructures showed that cellular damage due to ice crystal formation did not occur in the supercooled pineapple and agar gel, implying that the OMF successfully inhibited ice nucleation during supercooling. The weight losses of pineapple samples preserved with refrigeration, freezing, and supercooling for 14 days were determined to be 7.3%, 23.8%, and 0.8%, respectively. Furthermore, the visual appearance showed that supercooling effectively prolonged the shelf-life of pineapple without freezing damage. The results suggest that the developed OMF technology can inhibit ice nucleation during supercooling and can be used to preserve fresh-cut fruits at subzero temperature while ensuring the food quality. In addition, a solid model food could be used as a substitute for predicting the supercooling behavior of food products. Keywords: Food preservation, Fresh-cut fruit, Oscillating magnetic field, Pineapple, Supercooling.

105 COMPARISON BETWEEN TWO EXTENDERS FOR CRYOPRESERVATION OF BACTRIAN CAMEL SEMEN

2006 ◽  
Vol 18 (2) ◽  
pp. 161
Author(s):  
A. Niasari-Naslaji ◽  
S. Mosaferi ◽  
A. A. Gharahdaghi ◽  
A. Abarghani ◽  
A. Ghanbari ◽  
...  
Keyword(s):  
Citric Acid ◽  
Liquid Nitrogen ◽  
Egg Yolk ◽  
Deionized Water ◽  
Bactrian Camel ◽  
Equilibration Time ◽  
Persian Language ◽  
Significant Difference ◽  
Arcsine Transformation ◽  
Artificial Vagina

A Tris-based extender (SHOTOR diluent) has been developed for preserving Bactrian camel semen at 4�C (Niasari-Naslaji et al. 2005 Reprod. Fertil. Dev. 17, 198 (abstr.)). The present study investigated the possibility of utilizing the SHOTOR diluent for the cryopreservation of Bactrian camel semen. A modified bovine artificial vagina (Masaferi et al. 2005 Theriogeology 63, 92-101) was used to collect semen from three fertile bulls. The viscosity of the semen was reduced mechanically (Mosateri et al. 2005) and the homogenized semen was divided equally into two parts. Each part was sequentially diluted with either IMV buffers (Green buffer: first extender; White buffer: second extender; IMV, France) or SHOTOR diluents (without glycerol: first extender; with 12% glycerol: second extender). SHOTOR diluent consists of 2.6 g TIS, 1.35 g citric acid, 1.2 g glucose, and 0.9 g fructose in 100 mL of deionized water, with an osmolality of 330 mOsm/kg and pH of 6.9. All extenders had 20% egg yolk and antibiotics. The semen was diluted at the ratio of 1:1 with the first extender. The diluted semen was then cooled within 2 h to 4�C. At this temperature, the second extender was added at the same volume as the diluted semen in three steps with an equal volume, 10 min apart. After a 30-min equilibration time, beginning after addition of the last fraction of the second extender, the diluted semen was loaded into 0.5-mL straws at a concentration of 50 � 106 sperm per straw. The straws were maintained for 20 min at 4 cm above the liquid nitrogen surface, after which they were plunged into liquid nitrogen. The semen was thawed at 40�C water bath for 20 s. Progressive forward motility of spermatozoa was assessed at the time of dilution and immediately after thawing of the semen. The experiment was replicated four times. Data were analyzed using GLM procedure in SAS/STAT after arcsine transformation. At the time of dilution, there was no significant difference in progressive forward motility of spermatozoa between IMV buffers (51.8%) and SHOTOR diluent (61%; P > 0.05). However, after thawing, there was a significant decrease in progressive forward motility of spermatozoa in IMV buffers (4.2%) compared to SHOTOR diluent (29.9%, P < 0.05). In conclusion, in this experiment, SHOTOR diluent was more efficient for cryopreserving Bactrian camel semen than IMV extender. Shotor means camel in the Persian language.

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