Cryobiology

"Representations", N. 129, Winter 2015.

The essay analyzes the project of maintaining the body of V. I. Lenin in the Mausoleum in Moscow for the past ninety years, focusing on the unique biological science that developed around this project, and the unexpected political role this body has performed.

In this paper, we review and analyze the emerging network paradigm in organizational research. We begin with a conventional review of recent research organized around recognized research streams. Next, we analyze this research, developing a set of dimensions along which network studies vary, including direction of causality, levels of analysis, explanatory goals, and explanatory mechanisms. We use the latter two dimensions to construct a 2-by-2 table cross-classifying studies of network consequences into four canonical types: structural social capital, social access to resources, contagion, and environmental shaping. We note the rise in popularity of studies with a greater sense of agency than was traditional in network research.

Background The Depression Questionnaire (QD) is an instrument designed to measure depression in an inpatientrehabilitation setting. This study proposes a reduced version of the original 24-item QD, to make it better suited to the characteristics of patients in this setting. Methods The study population consisted of 801 individuals admitted to cardiac rehabilitation (564 males and 237 females) who had validly completed the original version of the QD. Data were analyzed first through confirmatory factor analysis and then using logistic models. Results Factor analysis of QD-24 items form, confirmed the plausibility of the monofactorial solution, both for males and females. Factor analysis combined with clinical observation suggested the possibility of reducing the QD to 17 items. Two further items were eliminated through use of simple logistic models. We thus obtained a shortened 15-item version of the QD (QD-R) that maintains a good index of separation (0.80), that is, good capacity to discriminate between individuals with different levels of depression, and a good item-trait interaction (v 2 153 = 165.37, P > 0.05). A second confirmatory factor analysis applied to the 15-item monofactorial model confirmed the validity of this shortened version of the instrument. The QD-R scores significantly correlated with metres walked during the 6 minute walking test (r = -0.316; P < 0.01). Conclusion Combined use of confirmatory factor analysis and simple logistic models together with observations drawn from clinical experience constitutes a valid method for shortening a questionnaire while at the same time maintaining, if not improving, its psychometric properties. The QD-R, seems adequate to how much is required in rehabilitation to point out the outcome, in case of variation of depression, easily to fill in by the debilitated individual or elderly, mainly because some item excessively influenced by the pathology or the hospital context were removed. Eur J Cardiovasc Prev Rehabil 00:000-000

Cryogelation is a physical hydrogel formation method for certain polymers, notably polyvinyl alcohol (PVA). The hypothesis of this study is that a PVA-based solution with the necessary intracellular cryoprotectant and nutrient supply can be used, first for storage of vascular smooth muscle cells, and subsequently to form a suitable tissue-engineering scaffold during the thawing process. Bovine arterial smooth muscle cells were encapsulated within PVA–gelatin hydrogels over a wide range of serum, DMSO and cell culture medium concentrations. Several parameters expected to affect gelation and cell viability (PVA viscosity, DMSO concentration, serum presence) were assessed with experimental designs and the optimal conditions for cell survival were determined. Cell viability can be improved by increasing concentration of DMSO and serum without compromising the gelation process. An additional crosslinking step using a coagulation bath was beneficial for hydrogel stability but caused peripheral accumulation of cells. In conclusion, a freeze–thaw process can be utilized to prepare and store cell-laden hydrogels with adjustable mechanical properties. Copyright © 2009 John Wiley & Sons, Ltd.

The ability to routinely cryopreserve micro-algal species reduces costs associated with maintaining large culture collections and reduces the risks of losing particular strains or species through contamination and genetic drift. Cryopreservation is also a useful adjunct in aquaculture hatcheries for strains of micro-algae where the nutritional status may change as a result of continuous sub-culture. In this study, cryopreservation of isolates from seven micro-algal classes was investigated. Successful candidates included the marine dinoXagellates Amphidinium carterae, Amphidinium trulla, and Gymnodinium simplex, and the haptophytes Chrysochromulina simplex, Prymnesium parvum, Prymnesium parvum f. patelliferum, Isochrysis galbana, and Pavlova lutheri. Also successfully cryopreserved were the planktonic diatoms Chaetoceros calcitrans, Chaetoceros muelleri, Chaetoceros sp., and the benthic Nitzschia ovalis, the chlorophyte Chlamydomonas coccoides, the rhodophyte Porphyridium purpureum, the prasinophytes Tetraselmis chuii, and Tetraselmis suecica, and the cyanophytes Raphidiopsis sp., and Aphanizomenon Xos-aquae. All species were successfully cryopreserved using 15% Me 2 SO. 

"Feeding people means producing population. Biotechnology, encompassing food production as well as assisted reproductive technology (ART), currently emerges as a most important apparatus (dispositif) of governing populations. It should be understood as a means of “biopower” because it not only contributes to reproducing life but also helps to improve and preserve it. Highly depending on refrigeration, modern biopower invents a new type of life, which is technologically self-sustained.

Therefore, sustainability is not only a question of “protecting the environment” but also of developing and maintaining an environment that allows us to dispose life: this is the cryogenic culture. In our paper, we trace the emergence and dissemination of what we call cryogenic life – meaning the ways of producing, distributing, maintaining and dispositioning organic matter via cooling, chilling and freezing. With the introduction of artificial coldness in the late 19th century and the expansion of the cold chain, these techniques have become a constitutive element of modern biopower.

Today, it seems that nearly every aspect of life is affected by cryogenic techniques: we cool our food, environments, drugs, organs, eggs, milk, semen, tissue, blood and much more. Our central argument is that these developments lead to the formation of a new form of life, which in many ways is the antipode of what Agamben calls bare life. In analysing the emergence of cryogenic culture from a biopower point of view, this study offers a new perspective on how populations are fostered and governed through regimes of freshness. While the history of chilled and frozen food slowly gains increasing attention in historical and cultural studies, the historical dynamics of the cryopolitical economy in the network society still need to be explored."

Oxidative stress significantly damages sperm functions such as motility, functional integrity, endogenous antioxidant enzyme activities and fertility due to lipid peroxidation induced by reactive oxygen species (ROS). The aim of this study was to determine the effects of antioxidants such as taurine and cysteine in Bioxcell Ò extender on standard semen parameters, fertilizing ability, lipid peroxidation (LPO) and antioxidant activities comprising reduced glutathione (GSH), glutathione peroxidase (GSH-Px), catalase (CAT) and superoxide dismutase (SOD) after the cryopreservation/thawing of bull semen. Nine ejaculates for each bull were included in the study. Three groups, namely taurine (2 mM), cysteine (2 mM), and control, were designed to analyze the antioxidants in Bioxcell Ò . Insemination doses were processed so that each 0.25-ml straw contained 15 Â 10 6 sperm.

A Cryopreservation of sperm cells is an essential process applied for long-term conservation of aquatic genetic resources. The goal of this research was to determine effect different of straw volumes and thawing rates on the post-thaw quality and fertilization ability of cryopreserved common carp (Cyprinus carpio) sperm. In this study, semen was cryopreserved according to conventional slow freezing protocol. For this aim, the cryosolution contained 75 mM NaCl, 70 mM KCl, 2 mM CaCl2, 1 mM MgSO4 and 20 mM Tris (pH: 8) supplemented with 10% MeOH. Following equilibration at +4 °C for 10 min, semen was packed into 0.25, 0.5 and 1.5 mL straws and frozen in liquid nitrogen vapour (for 10 min at-120 ºC) and finally stored in liquid nitrogen (-196 ºC) tank. Thawing of cryopreserved semen process was performed at 30 ºC for 10, 20 and 30 seconds in a water bath. Fertilization was performed using a ratio of 1x10 5 spermatozoa/egg. The highest fertility (68.4±2.5%) was determined with cryopreserved sperm packed in 1.5 mL straws that thawed at 30 ºC for 30 s. According to the results of this research, sperm cryopreserved with ionic extender containing 10% methanol and packed in 1.5 mL straws are suitable to achieve high fertilization of common carp eggs.

Terrestrially hibernating reptiles that live in seasonally cold climates need effective strategies of cold hardiness to survive the winter. Use of thermally buffered hibernacula is very important but when exposure to temperatures below 0°C cannot be avoided, either freeze avoidance (supercooling) or freeze tolerance strategies can be employed, sometimes by the same species depending on environmental conditions. Several reptile species display ecologically relevant freeze tolerance, surviving for extended times with 50% or more of their total body water frozen. The use of colligative cryoprotectants by reptiles is poorly developed but metabolic and enzymatic adaptations providing anoxia tolerance and antioxidant defense are important aids to freezing survival. New studies using DNA array screening are examining the role of freeze-responsive gene expression. Three categories of freeze responsive genes have been identified from recent screenings of liver and heart from freeze-exposed (5 h post-nucleation at À2.5°C) hatchling painted turtles, Chrysemys picta marginata. These genes encode (a) proteins involved in iron binding, (b) enzymes of antioxidant defense, and (c) serine protease inhibitors. The same genes were up-regulated by anoxia exposure (4 h of N 2 gas exposure at 5°C) of the hatchlings which suggests that these defenses for freeze tolerance are aimed at counteracting the injurious effects of the ischemia imposed by plasma freezing.

Current strategies for marine pollution monitoring are based on the integration of chemical and biological techniques. The sea urchin embryo-larval bioassays are among the biological methods most widely used worldwide. Cryopreservation of early embryos of sea urchins could provide a useful tool to overcome one of the main limitations of such bioassays, the availability of high quality biological material all year round. The present study aimed to determine the suitability of several permeant (dimethyl sulfoxide, Me 2 SO; propylene glycol, PG; and ethylene glycol, EG) and non-permeant (trehalose, TRE; polyvinylpyrrolidone, PVP) cryoprotectant agents (CPAs) and their combination, for the cryopreservation of eggs and embryos of the sea urchin Paracentrotus lividus. On the basis of the CPAs toxicity, PG and EG, in combination with PVP, seem to be most suitable for the cryopreservation of P. lividus eggs and embryos. Several freezing procedures were also assayed. The most successful freezing regime consisted on cooling from 4 to À12°C at 1°C/min, holding for 2 min for seeding, cooling to À20°C at 0.5°C/min, and then cooling to À35°C at 1°C/min. Maximum normal larvae percentages of 41.5% and 68.5%, and maximum larval growth values of 42.9% and 60.5%, were obtained for frozen fertilized eggs and frozen blastulae, respectively.

The goal of this study is to introduce the fundamental thermodynamic principles of isochoric (constant volume) cryopreservation for low temperature preservation of biological materials. Traditionally, cryopreservation is performed in an isobaric process (constant pressure) at 1 atm, because this is our natural environment and it is most convenient experimentally. More than half a century of studies on cryopreservation shows that the major mechanism of damage during isobaric cryopreservation is the increase in intracellular ionic concentration during freezing, which presumably causes chemical damage to the components of cells. Cryoprotectants as well as hyperbaric pressures have been developed as methods to reduce the extent of chemical damage during freezing. The theoretical studies in this paper show that in isochoric cryopreservation, the increase in solution concentration during freezing is lower at each temperature by almost an order of magnitude from that in isobaric cryopreservation. This suggests that isochoric cryopreservation could be a preferential alternative to isobaric cryopreservation. The technology for isochoric cryopreservation is very simple; freezing in a constant volume chamber. Using a simple isochoric cryopreservation device, we confirm the theoretical thermodynamic predictions.

The study was conducted to determine the influence of N-acetyl-L-cysteine (NAC) and superoxide dismutase (SOD) on chicken sperm motility, plasma membrane and acrosome integrity, mitochondrial activity, lipid peroxidation (LPO) and apoptotic changes after freezing-thawing process. Semen samples from fifteen Greenlegged Partridge roosters were pooled, diluted with EK extender without antioxidants (control), or supplemented with 5 mM NAC, or 200 U/mL SOD. Samples were subjected to cryopreservation. After thawing, sperm parameters evaluated by using CASA system and flow cytometry were assessed.

Background Physical activity (PA) and physical fitness (PF) are inversely associated with the clustering of cardiovascular disease (CVD) risk factors (RF) associated with the metabolic syndrome (MS). However, the role of individual components of PA, that is, intensity, duration and volume, and the inter-relationship with PF in the prevention of the MS and its individual components remains elusive. Design Cross-sectional analysis. Methods The study was based on 1298 (874 male and 424 female) police employees (aged 18-62 years) who participated in the Utrecht Police Lifestyle Intervention Fitness and Training study. PA was assessed with an extensive questionnaire. Peak oxygen uptake and metabolic markers, including blood pressure, fasting glucose, triglycerides, high-density lipoprotein cholesterol and waist circumference, were determined. Results The prevalence of the MS was 18.6% (22.5% in men, 10.6% in women). After adjustment for age and sex, average PA intensity, PA duration, PA volume and PF were each associated with reduced odds of MS. Regression analyses further showed an inverse relation between total CVD risk score and average PA intensity, the hours of PA performed at high intensity ( > 6 metabolic equivalent values) and PF, but no relation with total hours or the hours of PA performed at low or moderate intensity. When we adjusted our analyses for PF, the relations with the components of PA became nonsignificant. Using pathway analysis, we found that peak oxygen uptake mediated 78% of the effect of average PA intensity and 93% of the effect of the hours performed at high intensity on total CVD risk score. Conclusion PA and PF are inversely associated with the clustering of metabolic abnormalities. With regard to PA, it seems that intensity and more specifically higher intensity is the main characteristic of PA determining its effect on CVD RF. However, compared with PA, PF exerts greater effects on each of these individual CVD RF and its combination. Eur J Cardiovasc Prev Rehabil 16:677-683

Semen cryopreservation processes generally decrease sperm quality and fertility rate, but this is highly dependent on the specific susceptibility of sperm cells to low temperatures. In order to study the effect of cooling, freezing, and thawing on ram spermatozoa, sperm viability (plasma membrane integrity), HOS test (hyposmotic swelling test), and individual motility were assessed in fresh, cooled, and frozen-thawed samples. The cryoprotective ability of four extenders [Triladyl-yolk (20% Triladyl, 20% egg yolk), Salamon (9.9% raffinose, 2% sodium citrate, 15% egg yolk, 5% glycerol), I.N.I.A. (6.31% Tes, 1.51% Tris, 6% egg yolk, 3% glycerol), and Fiser (F 1 : 3.25% Tris, 9.3% fructose, 1.7% citric acid, 25% egg yolk, 2% glycerol; F 2 : 0.68% sodium citrate, 0.15% TES, 0.36% glycine, 10.18% lactose, 1.18% raffinose, 0.5% fructose, 3.95% dextran (150,000 -200,000 MW); 12% of the obtained solution was replaced by the same volume of glycerol)] was tested, as well as the effect of adding various compounds (bovine lactalbumin and serum albumin, vitamin E, dithiothreitol, bull seminal plasma, glycine betaine, proline, phosphatidylcholine, and cholesterol) to Fiser's medium. Simultaneously, centrifugal counter-current distribution in an aqueous two-phase system was carried out to analyze the effect of the four extenders on sperm surface heterogeneity. The results showed that ram spermatozoa undergo a dramatic loss of heterogeneity, viability, motility, and positive response to the HOS test during freezing and thawing. These changes were partially prevented by diluting samples in Fiser's extender containing egg yolk and glycerol. A considerable increase in sperm quality with respect to that obtained in Fiser's control sample was achieved by the addition of bovine lactalbumin, vitamin E, bovine serum albumin, and bovine and ram seminal plasma.

The Greenshell™ mussel (Perna canaliculus) is the main shellfish species farmed in New Zealand. The aim of this study was to evaluate the effects of cryoprotectant concentration, loading and unloading strategy as well as freezing and thawing method in order to develop a protocol for cryopreservation of trochophore larvae (16-20 h old). Toxicity tests showed that levels of 10-15% ethylene glycol (EG) were not toxic to larvae and could be loaded and unloaded in a single step. Through cryopreservation experiments, we designed a cryopreservation protocol that enabled 40-60% of trochophores to develop to D-larvae when normalized to controls. The protocol involved: holding at 0°C for 5 min, then cooling at 1°C min À1 to À10°C, holding for a further 5 min, then cooling at 0.5°C min À1 to À35°C followed by a 5 min hold and then plunging into liquid nitrogen. A final larval rearing experiment of 18 days was conducted to assess the ability of these frozen larvae to develop further. Results showed that only 2.8% of the frozen trochophores were able to develop to competent pediveligers.

The aim of this study was to evaluate the effects of ergothioneine and cysteamine as antioxidant supplements in a soybean lecithin extender for freezing ram semen. Twenty-four ejaculates were collected from four rams and diluted with extenders (1.5% soybean lecithin, 7% glycerol) containing no supplements (control) and cysteamine or ergothioneine (2, 4, 6 or 8mM). Motility by CASA, viability, plasma membrane functionality (HOS test), total abnormality, lipid peroxidation, glutathione peroxidase (GPx) activity and capacitation status (CTC staining) were assessed after thawing. Using 6mM of either antioxidant improved total motility. Cysteamine at 6mM and ergothioneine at 4 and 6mM improved viability and reduced lipid peroxidation (malondialdehyde concentration). Both antioxidants improved membrane functionality significantly, except at 8mM. Progressive motility, kinematic parameters, GPx activity, capacitation status and sperm abnormalities were not influenced by the antioxidant supp...

This study was conducted to determine the effects of methionine, inositol and carnitine on sperm (motility, abnormality, DNA integrity and in vivo fertility) and oxidative stress parameters (lipid peroxidation, total glutathione and antioxidant potential levels) of bovine semen after the freeze-thawing process. Nine ejaculates, collected with the aid of an artificial vagina twice a week from each Simmental bovine, were included in the study. Each ejaculate, splitted into seven equal groups and diluted in Tris-based extender containing methionine (2.5 and 7.5 mM), carnitine (2.5 and 7.5 mM), inositol (2.5 and 7.5 mM) and no additive (control), was cooled to 5°C and then frozen in 0.25 ml straws. Frozen straws were then thawed individually at 37°C for 20 s in a water bath for the evaluation.

The sea urchin has long been used as an invertebrate model organism in developmental
biology, membrane transport and sperm oocyte interactions, and for the assessment of marine
pollution. This review explores the effects of cryopreservation and biobanking in the biology
and development of sea urchins, all the way from germaplasm through to juveniles. This review
will provide an integral view of the process and all that is known so far about the biology of
cryopreserved sea urchins, as well as provide an insight on the applications of the biobanking of
these model organisms.

53233 rind Edwazls High Vacuum Limitetl, Manor Royal, Cruwley, Sussex, England To gather data concerning the thermal stabilities of preparations of interferon dried by sublimation of ice in vacua, the samples, in tip-seaIed grass ampoules, were placed in a water bath at 50°C for several months. Even though the ampoules which were tip-sealed in our laboratories tested negative by the methods used commonly for detecting "leakers" ( 1, 2), to our surprise many ampoules upon removal from the water bath contained beads of moisture on their interior surfaces.

The eastern oyster, Crassostrea virginica, is the most important cultured oyster species of the Atlantic and Gulf coasts of the United States. Cryopreservation of gametes and larvae of aquatic organisms has increased in importance in recent years. However, studies on the cryopreservation of sperm and larvae of mollusks have focused on the Pacific oyster, Crassostrea gigas. The present study was conducted to improve cryopreservation of sperm and trochophore larvae and to assess fertilizing ability and male-to-male variation of thawed sperm of the eastern oyster. Sperm were diluted in 12 cryoprotectant solutions composed of Hanks' balanced salt solution without calcium and 0, 5, 10, 15, 20, and 25% (v/v) propylene glycol with or without 0.25 M sucrose. Trochophore larvae were suspended in artificial seawater and 10 or 15% propylene glycol (v/v). Sperm or trochophore larvae were placed in 5-mL macrotubes and allowed to equilibrate for 15 min. The macrotubes were cooled in a controlledrate freezer at a rate of 2.5°C per min until reaching a final temperature of Ϫ30°C and were plunged into liquid nitrogen. After storage for 2 weeks, the samples were thawed in a water bath at 70°C for 15 s. Overall, for cryopreservation of sperm and larvae, best results were obtained using 10 or 15% propylene glycol. Thawed sperm presented significant male-to-male variation in fertilizing ability. Survival of thawed larvae decreased as the concentration of larvae per macrotube increased. The procedures developed in this study for sperm and larvae are suitable for production of seedstock in commercial oyster hatcheries. © 2001 Elsevier Science (USA)

Cryoprotectant toxicity is a fundamental limiting factor for the successful cryopreservation of living systems by both freezing and vitrification, and the ability to negate it would be attractive. Past attempts to demonstrate ''cryoprotectant toxicity neutralization" (CTN) have had many ups and downs. First convincingly introduced by Baxter and Lathe in 1971, the concept that certain amides can block toxic effects of dimethyl sulfoxide (Me 2 SO) was contradicted by direct experiments in 1990. But in 1995, the opposite mode of CTN, in which Me 2 SO blocked the damaging effects of formamide, was robustly demonstrated. Recent experiments have verified the original 1995 results and extended them to urea and acetamide, but no CTN was detected for N-methylamides (N-methylformamide, N,N-dimethylformamide, and N-methylacetamide). On the theory that the latter amides and acetamide might serve as low-toxicity structural analogs of formamide, urea, or Me 2 SO, competition experiments were carried out between them and formamide or urea, but CTN was not observed for these amide-amide systems. The idea that the N-methylamides might have non-specific rather than specific toxicity was supported by the fact that the concentrations of these amides that cause toxicity are similar to the concentrations that denature model proteins. Clear examples of neutralization of the toxicity of glycerol, propylene glycol, ethylene glycol, or Me 2 SO are presently lacking, but effects of the latter that depend on sulfhydryl oxidation have been reversed with reducing agents. In summary, CTN is a useful phenomenon with significant theoretical and practical implications.

Among the most widely used biological techniques in marine pollution assessment, the sea-urchin embryo-larval bioassay is in an advanced developmental stage. Cryopreservation might help to overcome the problem of the spawning seasonality and therefore strengthen the use of those embryo-larval bioassays. This work investigates different factors influencing cryopreservation of sea-urchin embryos, including the cooling rates and holding temperatures, the seeding, or the impact of plunging into liquid nitrogen. The blastula stage yielded better results than the fertilised egg, and the most effective cryoprotectants combination was dimethyl sulfoxide 1.5 M plus trehalose 0.04 M. The optimised protocol developed begins with an initial hold at 4°C for 2 min, followed by cooling at À1°C min À1 to À12°C. At this point a seeding step was incorporated with a 2 min hold, followed by a second cooling at À1°C min À1 to À80°C. After a final hold of 2 min the cryovials are transferred into liquid nitrogen for storage. Although the cryopreservation processes might cause a delay in the development of sea-urchin embryos, high larval growth (71-98% of controls) was obtained when cryopreserved blastulae were further incubated for 72-96 h in artificial seawater. We conclude that embryo-larval bioassays with cryopreserved sea-urchin blastulae are suitable for use in marine pollution monitoring programs and may be considered as an acceptable solution to the reproductive seasonality of sea-urchin species.

The recent decline and extinction of amphibian species is a worldwide phenomenon without an identified cause or solution. Assisted reproductive technologies, including sperm cryopreservation, are required to manage endangered amphibian species and preserve their genetic diversity. This study on the Anuran amphibian (Bufo marinus) was undertaken to determine the feasibility of cryopreservation of amphibian sperm. Sperm suspensions for cryopreservation were prepared by macerating testes in cryoprotective additives of 10% (w/v) sucrose or 10% (w/v) sucrose containing either 10, 15, or 20% (v/v) glycerol or 10, 15, or 20% (v/v) dimethyl sulfoxide (Me 2 SO). Suspensions were then cooled to Ϫ85°C using a controlled rate cooler, stored in LN 2 , and thawed in air. The motility and fertilization rate of cryopreserved suspensions and unfrozen control suspensions in Simplified Amphibian Ringer were compared. Sucrose alone had no cryoprotective effect. All other treatments showed varying degrees of recovery of motility and fertilizing capacity. High rates of recovery of motility and fertilizing capacity were observed with 15% Me 2 SO (68.9 Ϯ 3.8 and 60.5 Ϯ 4.7%) and 20% glycerol (58.0 Ϯ 5.9 and 81.4 Ϯ 4.3%), respectively. Motility and fertilization rates were similar with Me 2 SO but diverged with glycerol as cryoprotectant. The data demonstrate the feasibility of using sperm cryopreservation with amphibian species.

Delivery of cell therapies often requires the ability to hold products in readiness whilst logistical, regulatory and potency considerations are dealt with and recorded. This requires reversibly stopping biological time, a process which is often achieved by cryopreservation. However, cryo-preservation itself poses many biological and biophysical challenges to living cells that need to be understood in order to apply the low temperature technologies to their best advantage. This review sets out the history of applied cryopreservation, our current understanding of the various processes involved in storage at cryogenic temperatures, and challenges for robust and reliable uses of cryopreservation within the cell therapy arena.

The field of stem-cell biology has emerged as a key technology for the treatment of various disorders and tissue regeneration applications. However, a major problem remains in clinical practice, which is the question of whether stem cells preserve their self-renewal and differentiation potential in the culture conditions or not. In the current study, effects of boron on the cryopreservation of human tooth germ stem cells (hTGSCs) were evaluated for the first time. The impacts of various boron concentrations (sodium pentaborate pentahydrate (NaB)) were tested on characterized hTGSCs viability for different time intervals (24, 48, and 72 h). 20 lg/ml NaB with lower Me 2 SO concentration was found to display positive effects on hTGSCs during repeated freezing and defrosting cycles, and long-term cryopreservation. After thawing, cells were analyzed for their surface antigens and differentiation capacity. hTGSCs were successfully cryopreserved without any change in their mesenchymal stem cell characteristics as they were treated with boron containing freezing medium. In addition, fatty acid composition was examined to demonstrate membrane fatty acid profiles after freeze-thawing. Besides, NaB treatment extended osteogenic and chondrogenic differentiation of hTGSCs remarkably after long-term cryopreservation with respect to control groups. The study clearly suggests that NaB has a protective role on the survival of hTGSCs in short-and long-term cryopreservation. Due to the possible storage of hTGSCs at early ages, development of a functional and reliable cryopreservation media can be designed as a future solution to the dental stem cell banking.

Cryopreservation is widely used for long-term conservation of various tissues, embryos or gametes. However, few studies have described cryopreservation of invertebrate primary cell cultures and more particularly of marine invertebrate somatic cells. This technique would however be of great interest to facilitate the study of various metabolic processes which vary seasonally. The aim of the present study was to develop a protocol for cryopreservation of Crassostrea gigas vesicular cells. Different parameters were adjusted to improve recovery of cells after freezing. The most efficient cryoprotectant agent was a mix of Me 2 SO, glycerol, and ethylene glycol (4% each). The optimal cooling rate was −1 °C min −1 down to −70 °C before transfer into liquid nitrogen. In these conditions the percentage of viable cells reached 70% of the control. The glucose metabolism of thawed cells was evaluated using radioactive glucose as a tracer. Immediately after thawing, glucose uptake involving membrane transporters was greatly reduced (24% of control) whereas glucose incorporation into glycogen was less affected (68% of control).

The influence of various sugar types supplemented to the extender on post-thaw motility, viability, and fertilizing capacity of cryopreserved common carp (Cyprinus carpio) semen were investigated. The results indicated that types of sugar significantly influenced motility, motility duration, and viability rates (P<0.05). Glucose, maltose, sucrose, and trehalose provided higher motility compared to the sugar-free control in post-thaw samples. Trehalose provided highest progressive motility duration, and higher viable sperm rates were obtained with all sugar types except xylose. Xylose exhibited the lowest post-thaw progressive motility duration (35.2±1.4s). The mean highest fertilization (78.2±1.4%) and eyed egg rates (94.3±1.5%) were determined using trehalose in the extender, and differences between the treatments in the fertilization and eyeing rates were significant (P<0.05). Finally, the present study showed that sugars, especially maltose and trehalose (disaccharide), improved post-thaw spermatozoa motility and fertility in common carp semen. The IJA appears exclusively as a peer-reviewed on-line open-access journal at http://www.siamb.org.il/. To read papers free of charge, please register online at registration form. Sale of IJA papers is strictly forbidden.

The objectives of this study was to compare the effects of type and concentration of cryoprotectants glycerol (G), ethylene glycol (EG) and dimethyl sulfoxide (DMSO) on the plasma membrane and DNA integrity as well as antioxidant activity of cryopreserved Eastern Anatolian red bull sperm. Ejaculates were collected from the three bulls using an artificial vagina twice a week. The ejaculates were pooled to increase the semen volume for replication and to eliminate variability among the evaluated samples. The pooled ejaculates were also split into seven equal experimental groups and diluted with the modified base extender to a final spermatozoa concentration of 15 Â 10 6 /ml. The extended samples were cooled slowly to 4°C and equilibrated for 4 h. They were then loaded into 0.25 ml French straws and frozen using a digital freezing machine at 3 programmed rates: À3°C/min from +4°C to À10°C, À40°C/min from À10°C to À100°C, and À20°C/min from À100°C to À140°C. Thereafter, the straws were plunged into liquid nitrogen at À196°C. Frozen straws were thawed individually at 37°C for 30 s in a water bath to analyse progressive motility and sperm motion characteristics as well as membrane integrity using hypo-osmotic swelling test. Biochemical assays were performed in a spectrophotometer using commercial kits. DNA damage was evaluated by Comet Assay using Image Analysis System. 6% G exhibited the greatest percentages of CASA (43.7 ± 2.92%) and progressive (26.4 ± 2.64%) motilities when compared to the other groups (P < 0.001). 6% G and 6% EG showed the greatest values of preserved membrane integrity (P < 0.001). 6% DMSO and 3% EG + 3% DMSO resulted in greater chromatin damage than the other groups (P < 0.001). The antioxidant activities of GPx, GSH, and CAT as well as the total antioxidant activity were affected by the type of cryoprotectant; notably, 2% G + 2% EG + 2% DMSO yielded the lowest activities when compared to the other groups (P < 0.001).

The aim of current study was to evaluate effect of rosemary aqueous extract on post-thawed ram sperm quality in a soybean lecithin-based (SL) extender. Ram semen samples were obtained, extended with SL extender and supplemented with 0% (SL-R0), 2% (SL-R2), 4% (SL-R4), 6% (SL-R6), and 8% (SL-R8) rosemary aqueous extract. Following equilibration, the straws were frozen, and then plunged into the liquid nitrogen. After thawing, sperm motility and velocity parameters, plasma membrane functionality, viability, acrosomal and capacitation status were evaluated. Membrane lipid peroxidation was also analyzed through the malondialdehyde (MDA) concentration. Our results showed that SL-R4 and SL-R6 groups resulted in higher (p < 0.05) percentages of total motility, progressive motility, and plasma membrane functionality, as compared with other groups. Highest (p < 0.05) viable and lowest (p < 0.05) dead spermatozoa were observed in SL-R6 group compared to the other groups. The acrosomal and capacitation status were not affected (p > 0.05) by different levels of rosemary aqueous extract. Lower (p < 0.05) MDA concentration has been observed in SL-R4 and SL-R6 groups. The results of this study demonstrate that supplementation of SL extender with rosemary aqueous extract influences post-thawed ram sperm quality in a dose dependent manner.

Freeze-drying (Iyophilization) is a two-step dehydration process by which a substance is first frozen, and then water is removed by sublimation under vacuum. Freeze-drying has a diverse range of application, including the preparation of biological samples, pharmaceutical manufacture, and food preservation. Freeze-dried liquids show an abrupt, loss in product quality when the temperature of the subliming interface during drying is maintained above a critical value, t'he "collapse" temperature (6). Drying above this temperature gives a distorted (collapsed) product with poor solubility, low drying rates, uneven drying, and loss of texture and volatile substances. We find that, in the absence of solute crystallization, the collapse temperature depends principally upon the viscosity of the unfrozen port,ion of the solution. -4 simple theory, presentcd below, shows that increasing solute molecular weight gives higher collapse temperatures, and predict's observed collapse temperatures quantitatively.

Cryopreservation of boar semen is still considered suboptimal due to lower fertility when compared to fresh semen. The aim of this study was to evaluate the effects of the addition of different sugars (lactose, trehalose and glucose) on boar spermatozoa cryopreserved in an egg yolk based extender. Ejaculates were collected from a boar previously selected and semen samples were processed using the straw freezing procedure. In experiment 1, subsamples of semen were frozen in three different extenders: recommended lactose egg yolk extender (LEY); trehalose egg yolk extender (TEY) and glucose egg yolk extender (GEY). Sperm quality was assessed for motility, viability, acrosome integrity and hypoosmotic swelling test response upon collection, after freezing and thawing and then every hour for 3 h. Results showed that total motility at 1 and 3 h, progressive motility at 3 h, positive hypoosmotic response at 2 and 3 h and acrosome integrity at all times were significantly improved when trehalose was added to the extender. In experiment 2, sugar influence was also demonstrated in vitro fertilization. A total of 1691 oocytes were in vitro matured and inseminated with frozen-thawed sperm at 2000:1 sperm:oocyte ratio and coincubated for 6 h. Presumptive zygotes were cultured in NCSU-23 medium to assess fertilization parameters and embryo development. Both penetration and monospermy rates were significantly higher for trehalose frozen semen. A significant increase was observed in efficiency and blastocyst formation rates from TEY to the other groups. Our results demonstrated that trehalose extender enhances spermatozoa viability and its in vitro fertilization parameters in boar ejaculates with good sperm freezability. Further studies are necessary to assess the impact of sugars on the entire population.

Mutational inactivation of the genes designated DR1172 and DRB0118 in Deinococcus radiodurans R1 greatly sensitizes this species to desiccation, but not to ionizing radiation. These genes encode proteins that share features with the desiccation-induced LEA76 proteins of many plants and the PCC13-62 protein of Craterostigma plantagineum, suggesting that D. radiodurans may serve as a useful model for the study of desiccation tolerance in higher organisms.

We have carried out two experiments to study the cryobiology of red deer epididymal spermatozoa and to improve freezing extenders: (1) effect of extender (Tris-citrate-fructose) osmolality (300-600 mOsm/kg), and (2) effect of sugar (0.4 M) supplementation to the extender (no sugar, glucose, fructose, mannose, sucrose, maltose, threalose and raffinose). Sperm quality was assessed pre-freezing, post-thawing, and after 2 h at 37 8C post-thawing: sperm motility index (SMI), acrosome integrity and membrane integrity (HOS test) were assessed subjectively; mitochondrial activity (JC-1) and membrane stability (merocyanine 540) were assessed by flow cytometry. In experiment 2, DNA status was assessed using acridine orange and flow cytometry. To find an optimal osmolality, we fitted the data to a quadratic curve. Four hundred Osmolal per kilogram rendered better results prefreezing and post-thawing. However, post-thawing viability and most parameters after 2 h incubation fitted a linear model. Osmolalities above 425 mOsm/kg were deleterious (P < 0.05). In experiment 2, fructose had a positive effect respect to control after 2 h of incubation at 37 8C post-thawing. Di and trisaccharides were deleterious. Trehalose showed impaired DNA status after 2 h incubation. In conclusion, the osmolality of the extenders should be around 400 mOsm/kg, although the change from quadratic to lineal may indicate a complex effect that must be further studied. Monosaccharides may enhance red deer epididymal sperm cryopreservation, especially fructose, whereas di and trisaccharides may not be adequate. #

The determination of the space-or time-dependent heat transfer coefficient which links the boundary temperature to the heat flux through a third-kind Robin boundary condition in transient heat conduction is investigated. The reconstruction uses average surface temperature measurements. In both cases of the space-or time-dependent unknown heat transfer coefficient the inverse problems are nonlinear and ill posed. Least-squares penalized variational formulations are proposed and new formulae for the gradients are derived. Numerical results obtained using the nonlinear conjugate gradient method combined with a boundary element direct solver are presented and discussed.

Coral species throughout the worldÕs oceans are facing severe environmental pressures. We are interested in conserving coral larvae by means of cryopreservation, but little is known about their cellular physiology or cryobiology. These experiments examined cryoprotectant toxicity, dry weight, water and cryoprotectant permeability using cold and radiolabeled glycerol, spontaneous ice nucleation temperatures, chilling sensitivity, and settlement of coral larvae. Our two test species of coral larvae, Pocillopora damicornis (lace coral), and Fungia scutaria (mushroom coral) demonstrated a wide tolerance to cryoprotectants. Computer-aided morphometry determined that F. scutaria larvae were smaller than P. damicornis larvae. The average dry weight for P. damicornis was 24.5%, while that for F. scutaria was 17%, yielding osmotically inactive volumes (V b ) of 0.22 and 0.15, respectively. The larvae from both species demonstrated radiolabeled glycerol uptake over time, suggesting they were permeable to the glycerol. Parameter fitting of the F. scutaria larvae data yielded a water permeability P2 lm/min/atm and a cryoprotectant permeability = 2.3 · 10 À4 cm/min while modeling indicated that glycerol reached 90% of final concentration in the larvae within 25 min. The spontaneous ice nucleation temperature for F. scutaria larvae in filtered seawater was À37.8 ± 1.4°C. However, when F. scutaria larvae were chilled from room temperature to À11°C at various rates, they exhibited 100% mortality. When instantly cooled from room temperature to test temperatures, they showed damage below 10°C. These data suggest that they are sensitive to both the rate of chilling and the absolute temperature, and indicate that vitrification may 0011-2240/$ -see front matter Ó

Survival of buck sperm is affected due to duration and temperature of stages of refrigerated or frozen storage. This study investigated interactive effect of cooling rates (moderate; MC and rapid cooling; RC); and equilibration times (0, 2, 4 and 8 h) on survival before freezing at 4°C and post-thaw quality of buck sperm. Semen was collected (three Beetal bucks; replicates = 6), pooled and diluted with Triscitrate extender. Pooled semen samples were subjected to either RC (À2.2°C/min) or MC (À0.3°C/min) from 37°C to 4°C in separate aliquots and further equilibrated at 4°C for 8 h. Semen was frozen using standard procedure after completion of each equilibration period i.e. 0, 2, 4 and 8 h. Semen was evaluated for motility, viability, plasma membrane integrity (PMI) and normal apical ridge (NAR) before freezing and after thawing. The survival time (time for survival above threshold limit i.e. 60%) at 4°C, of motility and PMI was observed 5 and 6 h respectively in RC group while >8 h in MC group. Rate of decline (slope) in motility and viability was higher (P < 0.05) in RC overtime during equilibration at 4°C while PMI and NAR declined at equal rate in both cooling groups. Post-thaw motility and NAR were higher (P < 0.05) in MC when equilibrated for 2-8 h while viability and PMI of RC was observed equal to MC group. In conclusion, survival of buck sperm is higher when cooled with moderate rate. However, RC can maintain post-thaw sperm viability and PMI equal to MC when equilibrated for 2-8 h. The methods should be explored to maintain motility and NAR during rapid cooling of buck sperm.