Investigating the deep supercooling ability of an Alaskan beetle, Cucujus clavipes puniceus, via high throughput proteomics

Cucujus clavipes puniceus is a freeze avoiding beetle capable of surviving the long, extremely cold winters of the Interior of Alaska. Previous studies showed that some individuals typically supercool to mean values of approximately − 40 °C, with some individuals supercooling to as low as − 58 °C, but these non-deep supercooling (NDSC) individuals eventually freeze if temperatures drop below this. However, other larvae, especially if exposed to very cold temperatures, supercool even further. These deep supercooling (DSC) individuals do not freeze even if cooled to − 100 °C. In addition, the body water of the DSC larvae vitrifies (turns to a glass) at glass transition temperatures of − 58 to − 70 °C. This study examines the proteomes of DSC and NDSC larvae to assess proteins that may contribute to or inhibit the DSC trait. Using high throughput proteomics, we identified 138 proteins and 513 Gene Ontology categories in the DSC group and 104 proteins and 573 GO categories in the NDSC group. GO categories enriched in DSC include alcohol metabolic process, cellular component morphogenesis, monosaccharide metabolic process, regulation of biological quality, extracellular region, structural molecule activity, and antioxidant activity. Proteins unique to DSC include alpha casein precursor, alpha-actinin, vimentin, tropomyosin, beta-lactoglobulin, immunoglobulins, tubulin, cuticle proteins and endothelins.     

Biodegradable starch based nanocomposites with low water vapor permeability and high storage modulus

Nanocomposite materials based on a starch matrix reinforced with very small amounts of multi-walled carbon nanotubes (MWCNTs) (from 0.005 wt% to 0.055 wt%) were developed. The material's dynamic-mechanical and water vapor permeability properties were investigated. An increasing trend of storage modulus (E′) and a decreasing trend of water vapor permeability (WVP) with filler content were observed at room temperature. For the composite with 0.055 wt% of filler, E′ value was about 100% higher and WVP value was almost 43% lower than the corresponding matrix values. MWCNTs were wrapped in an aqueous solution of a starch-iodine complex before their incorporation into the matrix, obtaining exceptionally well-dispersed nanotubes and optimizing interfacial adhesion. This excellent filler dispersion leads to the development of an important contact surface area with the matrix material, producing remarkable changes in the starch-rich phase glass transition temperature even in composites with very low filler contents. This transition is shifted towards higher temperatures with increasing content of nanotubes. So at room temperature, some composites are in the rubber zone while others, in the transition zone. Therefore, this change in the material glass transition temperature can be taken as responsible for the important improvements obtained in the composites WVP and E′ values for carbon nanotubes content as low as 0.05 wt%.     

Thermal properties of agave fructans (Agave tequilana Weber var. Azul)

Thermal properties of agave (A. tequilana Weber var. Azul) at different water contents were investigated. HP-TLC results showed a complex mixture of mono-, di-, oligo, and polysaccharides in agave fructans samples. The thermal decomposition temperatures were observed below to 200 °C. Modulated-differential scanning calorimetry studies showed a glass transition and a relaxation enthalpy processes in agave fructans. Samples with the highest moieties of monosaccharides showed the lower glass transition temperatures (Tg). The moisture sorption isotherm of agave fructans was determined at 20 °C and fitted to the GAB model. Gordon-Taylor equation was used to fit the Tg experimental data as a function of water content. Agave fructans was found to be an amorphous material. At low water activity (a_w) values (<0.4), agave fructans remained in a powdered amorphous state; and at intermediate a_w (0.4-0.75) collapsed and caked; and at high a_w (>0.75) changed in a highly viscous liquid-like solution.     

Dynamics of uncrystallized water and protein in hydrated elastin studied by thermal and dielectric techniques

Dynamics of uncrystallized water and protein was studied in hydrated pellets of the fibrous protein elastin in a wide hydration range (0 to 23 wt.%), by differential scanning calorimetry (DSC), thermally stimulated depolarization current technique (TSDC) and dielectric relaxation spectroscopy (DRS). Additionally, water equilibrium sorption–desorption measurements (ESI) were performed at room temperature. The glass transition of the system was studied by DSC and its complex dependence on hydration water was verified. A critical water fraction of about 18 wt.% was found, associated with a reorganization of water in the material. Three dielectric relaxations, associated to dynamics related to distinct uncrystallized water populations, were recorded by TSDC and DRS. The low temperature secondary relaxation of hydrophilic polar groups on the protein surface triggered by hydration water for almost dry samples contains contributions from water molecules themselves at higher water fractions (ν relaxation). This particular relaxation is attributed to water molecules in the primary and secondary hydration shells of the protein fibers. At higher temperatures and for water fraction values equal to or higher than 10 wt.%, a local relaxation of water molecules condensed within small openings in the interior of the protein fibers was recorded. The evolution of this relaxation (w relaxation) with hydration level results in enhanced cooperativity at high water fraction values, implying the existence of “internal” water confined within the protein structure. At higher temperatures a relaxation associated with water dynamics within clusters between fibers (p relaxation) was also recorded, in the same hydration range.     

Stabilization of Na,K-ATPase by ionic interactions

The effect of ions on the thermostability and unfolding of Na,K-ATPase from shark salt gland was studied and compared with that of Na,K-ATPase from pig kidney by using differential scanning calorimetry (DSC) and activity assays. In 1 mM histidine at pH 7, the shark enzyme inactivates rapidly at 20 °C, as does the kidney enzyme at 42 °C (but not at 20 °C). Increasing ionic strength by addition of 20 mM histidine, or of 1 mM NaCl or KCl, protects both enzymes against this rapid inactivation. As detected by DSC, the shark enzyme undergoes thermal unfolding at lower temperature (T_m ≈ 45 °C) than does the kidney enzyme (T_m ≈ 55 °C). Both calorimetric endotherms indicate multi-step unfolding, probably associated with different cooperative domains. Whereas the overall heat of unfolding is similar for the kidney enzyme in either 1 mM or 20 mM histidine, components with high mid-point temperatures are lost from the unfolding transition of the shark enzyme in 1 mM histidine, relative to that in 20 mM histidine. This is attributed to partial unfolding of the enzyme due to a high hydrostatic pressure during centrifugation of DSC samples at low ionic strength, which correlates with inactivation measurements. Addition of 10 mM NaCl to shark enzyme in 1 mM histidine protects against inactivation during centrifugation of the DSC sample, but incubation for 1 h at 20 °C prior to addition of NaCl results in loss of components with lower mid-point temperatures within the unfolding transition. Cations at millimolar concentration therefore afford at least two distinct modes of stabilization, likely affecting separate cooperative domains. The different thermal stabilities and denaturation temperatures of the two Na,K-ATPases correlate with the respective physiological temperatures, and may be attributed to the different lipid environments.     

Activity patterns in northern rock sole are mediated by temperature and feeding history

Studies of activity patterns of fishes rarely consider the impact of environmental conditions or organism state. In this paper we demonstrate the influence of temperature and feeding history on the diel activity patterns of juvenile (age 1+) northern rock sole Lepidopsetta polyxystra (Orr and Matarese). Activity type (benthic vs. water column) and level were determined from hourly video recordings of fish in laboratory tanks with the aid of infrared illumination. Groups of rock sole (n = 4 per group) were observed at 2, 5, 9 and 13 °C without food and at 9 °C with food offered once in the morning, at dusk or at night. In all experiments, rock sole exhibited peak activity levels during the dusk and dawn transition periods and a temporal segregation of activity mode. Daytime and crepuscular activity was predominantly benthic, occurring within a few centimeters of the tank bottom, whereas a significant fraction of nighttime activity (up to 90%) occurred with rock sole swimming in the water column or at the water surface. The primary effect of water temperature on rock sole behavior was a reduction in daytime and crepuscular activity at low temperatures. Conversely, nighttime activity levels were independent of water temperature, resulting in a thermally induced shift in the primary activity period. Morning and dusk feeding produced a short-term (1 h) elevation in activity followed by activity suppression until the subsequent crepuscular phase. Nighttime feeding resulted in a longer period of elevated activity (2 h) and did not suppress further benthic or water column activity. These observations suggest a temporal segregation of activity between daytime foraging and nighttime movement that balances energetic demands with predation risk.     

Temperature and pressure effects on structural and conformational properties of POPC/SM/cholesterol model raft mixtures—a FT-IR, SAXS, DSC, PPC and Laurdan fluorescence spectroscopy study

We report on the effects of temperature and pressure on the structure, conformation and phase behavior of aqueous dispersions of the model lipid “raft” mixture palmitoyloleoylphosphatidylcholine (POPC)/bovine brain sphingomyelin (SM)/cholesterol (Chol) (1:1:1). We investigated interchain interactions, hydrogen bonding, conformational and structural properties as well as phase transformations of this system using Fourier transform-infrared (FT-IR) spectroscopy, small-angle X-ray scattering (SAXS), differential scanning calorimetry (DSC) coupled with pressure perturbation calorimetry (PPC), and Laurdan fluorescence spectroscopy. The IR spectral parameters in combination with the scattering patterns from the SAXS measurements were used to detect structural and conformational transformations upon changes of pressure up to 7-9 kbar and temperature in the range from 1 to about 80 °C. The generalized polarization function (GP) values, obtained from the Laurdan fluorescence spectroscopy studies also reveal temperature and pressure dependent phase changes. DSC and PPC were used to detect thermodynamic properties accompanying the temperature-dependent phase changes. In combination with literature fluorescence spectroscopy and microscopy data, a tentative p,T stability diagram of the mixture has been established. The data reveal a broad liquid-order/solid-ordered (l_o + s_o) two-phase coexistence region below 8 ± 2 °C at ambient pressure. With increasing temperature, a l_o + l_d + s_o three-phase region is formed, which extends up to ∼27 °C, where a liquid-ordered/liquid-disordered (l_o + l_d) immiscibility region is formed. Finally, above 48 ± 2 °C, the POPC/SM/Chol (1:1:1) mixture becomes completely fluid-like (liquid-disordered, l_d). With increasing pressure, all phase transition lines shift to higher temperatures. Notably, the l_o + l_d (+s_o) phase coexistence region, mimicking raft-like lateral phase separation in natural membranes, extends over a rather wide temperature range of about 40 °C, and a pressure range, which extends up to about 2 kbar for T = 37 °C. Interestingly, in this pressure range, ceasing of membrane protein function in natural membrane environments has been observed for a variety of systems.     

Determination of the oxidative stability by DSC of vegetable oils from the Amazonian area

Differential scanning calorimetry (DSC) and a Rancimat method apparatus were applied to evaluate the oxidative stability of buriti pulp oil (Mauritia flexuosa Mart), rubber seed oil (Hevea brasiliensis), and passion fruit oil (Passiflora edulis). The Rancimat measurements taken for the oxidative induction times were performed under isothermal conditions at 100 °C and in an air atmosphere. The DSC technique involved the oxidation of oil samples in an oxygen-flow DSC cell. The DSC cell temperature was set at five different isothermal temperatures: 100, 110, 120, 130 and 140 °C. During the oxidation reaction, an increase in heat was observed as a sharp exothermic curve. The value T₀ represents the oxidative induction time, which is determined from the downward extrapolated DSC oxidative curve verses the time axis. These curves indicate a good correlation between the DSC T₀ and oxidative stability index (OSI) values. The DSC method is useful because it consumes less time and less sample.     

Glass transition behavior of the vitrification solutions containing propanediol, dimethyl sulfoxide and polyvinyl alcohol

Knowledge of the glass transition behavior of vitrification solutions is important for research and planning of the cryopreservation of biological materials by vitrification. This brief communication shows the analysis for the glass transition and glass stability of the multi-component vitrification solutions containing propanediol (PE), dimethyl sulfoxide (Me₂SO) and polyvinyl alcohol (PVA) by using differential scanning calorimetry (DSC) during the cooling and subsequent warming between 25 and −150 °C. The glass formation of the solutions was enhanced by introduction of PVA. Partial glass formed during cooling and the fractions of free water in the partial glass matrix increased with the increasing of PVA concentration, which caused slight decline of glass transition temperature, T_g. Exothermic peaks of devitrification were delayed and broadened, which may result from the inhibition of ice nucleation or recrystallization of PVA.     

Effect of desiccation level and storage temperature on green spore viability of Osmunda japonica

In order to effectively preserve green spores, which have relatively higher water content and lose viability more quickly than non-green spores, we studied the effect of desiccation level and storage temperature on Osmunda japonica spores. The water content of fresh spores was 11.20%. After 12 h desiccation by silica gel, the water content decreased to 6% but spore viability did not change significantly. As the desiccation continued, the decrease in water content slowed, but spore viability dropped. For almost all storage periods, the effects of storage temperature, desiccation level, and temperature × desiccation level were significantly different. After seven days of storage, spores at any desiccation level stored at 4 °C obtained high germination rates. After more than seven days storage, liquid nitrogen (LN) storage obtained the best results. Storage at −18 °C led to the lowest germination rates. Spores stored at room temperature and −18 °C all died within three months. For storage at 4 °C and in LN, spores desiccated 12 and 36 h obtained better results. Spores without desiccation had the highest germination rates after being stored at room temperature, but suffered the greatest loss after storage at −18 °C. These results suggest that LN storage is the best method of long-term storage of O. japonica spores. The critical water content of O. japonica spores is about 6% and reduction of the water content to this level improves outcome after LN storage greatly. The reason for various responses of O. japonica spores to desiccation and storage temperatures are discussed.     

Effects of freezing on membranes and proteins in LNCaP prostate tumor cells

Fourier transform infrared spectroscopy (FTIR) and cryomicroscopy were used to define the process of cellular injury during freezing in LNCaP prostate tumor cells, at the molecular level. Cell pellets were monitored during cooling at 2 °C/min while the ice nucleation temperature was varied between − 3 and − 10 °C. We show that the cells tend to dehydrate precipitously after nucleation unless intracellular ice formation occurs. The predicted incidence of intracellular ice formation rapidly increases at ice nucleation temperatures below − 4 °C and cell survival exhibits an optimum at a nucleation temperature of − 6 °C. The ice nucleation temperature was found to have a great effect on the membrane phase behavior of the cells. The onset of the liquid crystalline to gel phase transition coincided with the ice nucleation temperature. In addition, nucleation at − 3 °C resulted in a much more co-operative phase transition and a concomitantly lower residual conformational disorder of the membranes in the frozen state compared to samples that nucleated at − 10 °C. These observations were explained by the effect of the nucleation temperature on the extent of cellular dehydration and intracellular ice formation. Amide-III band analysis revealed that proteins are relatively stable during freezing and that heat-induced protein denaturation coincides with an abrupt decrease in α-helical structures and a concomitant increase in β-sheet structures starting at an onset temperature of approximately 48 °C.     

Effect of water temperature on dietary protein requirement,growth and body composition of Asian catfish, Clarias batrachus fry

This study aimed to evaluate the protein requirement of Clarias batrachus fry, were estimated at two different water temperatures, 28 and 32 °C. The influence of dietary protein level and water temperature on body composition, weight gain, food and nutrient utilization were estimated. The Asian catfish, C. batrachus fry were fed four diets containing 28% (diet 1), 32% (diet 2), 36% (diet 3) and 40% (diet 4) protein levels and reared at two water temperatures 28 and 32 °C for 60 days. Fry fed with diet 3 containing 36% protein showed the highest mean final body weight at 32 °C. Final body weight was significantly (P<0.05) affected by dietary treatments and temperatures. Clarias batrachus fry raised at 28 °C had higher feed efficiency (93.20%) than the fry reared at 32 °C (87.58%) with 28% dietary protein level. Further, feed efficiency decreased with increase in dietary protein level. Higher daily protein retention (0.089%) observed at lower (0.0217 g) daily protein intake at 28 °C than 0.0283 g at 32 °C. While, optimal (0.0282 g) daily protein intake showed higher daily weight gain at 32 °C. Productive protein value (% PPV) was maximum (1.76%) at 32 °C than at 28 °C (0.76%). Final body lipid recorded higher value than initial body lipid at both the temperatures. Hepatosomatic index (HSI) observed to have been influenced (P<0.05) by diets and temperatures, while viscerosomatic index (VSI) affected (P<0.05) by only diets and not (P>0.05) by temperatures. The study concluded that the diet 3 containing 36% protein was optimal for growth of C. batrachus fry at both the temperatures.     

Thermal tolerance of Litopenaeus vannamei (Crustacea: Penaeidae) acclimated to four temperatures

Critical thermal minima (CTMin) and maxima (CTMax) values were determined for the Pacific white shrimp Litopenaeus vannamei post-larvae and juveniles at four different acclimation temperatures (15, 20, 25, and 30 °C). The CTMin of shrimp at these acclimation temperatures were 7.82, 8.95, 9.80, and 10.96 °C for post-larvae and 7.50, 8.20, 10.20, and 10.80 °C for juveniles, respectively, at 1 °C h⁻¹ cooling rate. The CTMax values were 35.65, 38.13, 39.91, and 42.00 °C for post-larvae and 35.94, 38.65, 40.30, and 42.20 °C for juveniles at the respective acclimation temperatures. Both acclimation temperature and size of the shrimp affected CTMin values of L. vannamei (P<0.01). Overall, juveniles displayed significantly lower CTMin values than the post-larvae (P<0.0001). However, the CTMax response by post-larvae and juveniles were not significantly different from each other and no interaction was determined between the acclimation temperature and development stage (P>0.01). The area of the thermal tolerance polygon over four acclimation temperatures (15, 20, 25, and 30 °C) for the post-larvae of L. vannamei was calculated to be 434.94 °C². The acclimation response ratio (ARR) values were high ranging from 0.35 to 0.44 for both post-larvae and juveniles. L. vannamei appears to be more sensitive to low temperatures than other penaeid species and its cold tolerance zone ranged from 7.5 to 11 °C. In successful aquaculture temperature must never fall below 12 °C to prevent mortalities. Upper thermal tolerance is less of a problem as in most subtropical regions maximum water temperature rarely exceeds 34 °C, but care should be given if shallow ponds with low water renewal rate are being used.     

Calorimetric measurement of water transport and intracellular ice formation during freezing in cell suspensions

The current study presents a new and novel analysis of heat release signatures measured by a differential scanning calorimeter (DSC) associated with water transport (WT), intracellular ice formation (IIF) and extracellular ice formation (EIF). Correlative cryomicroscopy experiments were also performed to validate the DSC data. The DSC and cryomicroscopy experiments were performed on human dermal fibroblast cells (HDFs) at various cytocrit values (0–0.8) at various cooling rates (0.5–250 °C/min). A comparison of the cryomicroscopy experiments with the DSC analysis show reasonable agreement in the water transport (cellular dehydration) and IIF characteristics between both the techniques with the caveat that IIF measured by DSC lagged that measured by cryomicroscopy. This was ascribed to differences in the techniques (i.e. cell vs. bulk measurement) and the possibility that not all IIF is associated with visual darkening. High and low rates of 0.5 °C/min and 250 °C/min were chosen as HDFs did not exhibit significant IIF or WT at each of these extremes respectively. Analysis of post-thaw viability data suggested that 10 °C/min was the presumptive optimal cooling rate for HDFs and was independent of the cytocrit value. The ratio of measured heat values associated with IIF (q_IIF) to the total heat released from both IIF and water transport or from the total cell water content in the sample (q_CW) was also found to increase as the cooling rate was increased from 10 to 250 °C/min and was independent of the sample cytocrit value. Taken together, these observations suggest that the proposed analysis is capable of deconvolving water transport and IIF data from the measured DSC latent heat thermograms in cell suspensions during freezing.     

Thermo-sensitivity and triggered drug release of polysaccharide nanogels derived from pullulan-g-poly(l-lactide) copolymers

Thermo-responsive nanogels from poly(l-lactide)-g-pullulan (PLP1 and 2) copolymers with different lactide contents were investigated as an anticancer drug delivery carrier. The phase transition temperature of PLP 1 with lower lactide content in distilled water showed around 35 °C. Upon adding 0.15 M NaCl to PLP 1, a significant difference in the transmittance was observed when comparing the non-additive salt condition. The total amount of released doxorubicin (DOX) from the DOX-loaded PLP nanogels increased with increasing temperature for 50 h. A noticeable difference in the initial release by PLP 1 was observed between 37 and 42 °C. In the 50% inhibitory concentration (IC₅₀) analysis, the IC₅₀ values of DOX released from PLP 1 were approximately 5.9 and 9.3 μg/mL at 37 and 42 °C, respectively. The results suggest that self-assembled PLP nanogels, by means of a triggering temperature, can be used as a long-term drug delivery system in cancer treatments.     

Thermogenic characteristics and evaporative water loss in the tree shrew (Tupaia belangeri)

Thermogenic characteristics and evaporative water loss were measured at different temperatures in Tupaia belangeri. The thermal neutral zone (TNZ) of T. belangeri was 30–35 °C. Mean body temperature was 39.76±0.27 °C and mean body mass was 100.86±9.09 g. Basal metabolic rate (BMR) was 1.38±0.03 ml O₂/g h. Average minimum thermal conductance (C_m) was 0.13±0.01 ml O₂/g h °C. Evaporative water loss in T. belangeri increased when the temperature rose; the maximal evaporative water loss was 3.88±0.41 mg H₂O/g h at 37.5 °C. The results may reflect features of small mammals in the sub-tropical plateau region: T. belangeri had high basal metabolic rate and high total thermal conductance, compared with the predicted values based on their body mass whilst their body temperatures are relatively high; T. belangeri has high levels of evaporative water loss and poor water-retention capacity. Evaporative water loss plays an important role in temperature regulation.     

Effect of thermal pretreatment on equilibrium moisture content of lignocellulosic biomass

The equilibrium moisture content (EMC) of raw lignocellulosic biomass, along with four samples subjected to thermal pretreatment, was measured at relative humidities ranging from 11% to 97% at a constant temperature of 30 °C. Three samples were prepared by treatment in hot compressed water by a process known as wet torrefaction, at temperatures of 200, 230, and 260 °C. An additional sample was prepared by dry torrefaction at 300 °C. Pretreated biomass shows EMC below that of raw biomass. This indicates that pretreated biomass, both dry and wet torrefied, is more hydrophobic than raw biomass. The EMC results were correlated with a recent model that takes into account additional non-adsorption interactions of water, such as mixing and swelling. The model offers physical insight into the water activity in lignocellulosic biomass.     

Partitioning of transpiratory water loss of the desert scorpion, Hadrurus arizonensis (Iuridae)

Terrestrial arthropods lose body water to the environment mainly through transpiration. The aim of this study was to determine the fraction of respiratory losses from total transpiratory water loss in scorpions, as relatively high respiratory losses would indicate a fitness benefit from regulation of gas-exchange rate under stressful desiccating conditions. We measured metabolic rates and water-loss rates of Hadrurus arizonensis (Iuridae) at a range of ecologically-relevant temperatures. Calculation of respiratory water losses was based on increased metabolic and water-loss rates during nocturnal activity (assuming no change in cuticular resistance at a given constant experimental temperature). Respiratory losses accounted for 9.0 ± 1.7% of total transpiratory losses at 25 °C, doubled to 17.9 ± 1.8% at 30 °C and increased to 31.0 ± 2.0% at 35 °C (n = 5, 15 and 15, respectively). Furthermore, the relative importance of respiratory transpiration is likely to be higher at temperatures above 35 °C, which have been recorded even within the burrows of H. arizonensis. Measurements of cuticular lipid melting points do not provide evidence for increased cuticular resistance to water loss at higher temperatures. However, the relatively high fraction of respiratory water losses reported here for H. arizonensis supports the notion of respiratory regulation as an evolved mechanism for conserving scorpion body water stores under stressful conditions.     

Pre-freeze mortality in three species of aphids from sub-Antarctic Marion Island

Understanding the mechanisms by which aphids survive low temperature is fundamental in forecasting the risk of pest outbreaks. Aphids are chill susceptible and die at a temperature close to that at which a small exothermal event is produced. This event, which can be identified using differential scanning calorimetry (DSC), normally occurs at a higher temperature than the supercooling point (SCP) and has been termed a pre-freeze event (PFE). However, it is not known what causes the PFE or whether it signifies the death of the aphid. These questions are addressed here by using a sensitive DSC to quantify the PFE and SCP and to relate these thermal events to the lower lethal temperature (LT₅₀) of sub-Antarctic aphids acclimated to low temperatures. PFEs were observed in each of the 3 species of aphids examined. They occurred over a narrower temperature range and at a higher temperature range than the SCP (−8.2 to −13.8 and −5.6 to −29.8 °C, respectively). Increased acclimation temperature resulted in increased SCPs in Myzus ascalonicus but not in Rhopalosiphum padi. The LT₅₀ reduced by approximately 1 °C from −9.3 to −10.5 °C with reduced acclimation temperature (10–0 °C). The LT₅₀ was close to the temperature at which the PFE occurred but statistically significantly higher than either the PFE or the SCP. In the majority of cases the PFE exotherm occurred well before the main exotherm produced by the bulk of the insect’s body water freezing (SCP). However, in a few cases it occurred at the same temperature or before the super-cooling point making the term, pre-freeze event (PFE), rather misleading. The possible origins of the PFE are discussed.     

Implications of storage and handling conditions on glass transition and potential devitrification of oocytes and embryos

Devitrification, the process of crystallization of a formerly crystal-free, amorphous glass state, can lead to damage during the warming of cells. The objective of this study was to determine the glass transition temperature of a cryopreservation solution typically used in the vitrification, storage, and warming of mammalian oocytes and embryos using differential scanning calorimetry. A numerical model of the heat transfer process to analyze warming and devitrification thresholds for a common vitrification carrier (open-pulled straw) was conducted. The implications on specimen handling and storage inside the dewar in contact with nitrogen vapor phase at different temperatures were determined. The time required for initiation of devitrification of a vitrified sample was determined by mathematical modeling and compared with measured temperatures in the vapor phase of liquid nitrogen cryogenic dewars. Results indicated the glass transition ranged from −126 °C to −121 °C, and devitrification was initiated at −109 °C. Interestingly, samples entered rubbery state at −121 °C and therefore could potentially initiate devitrification above this value, with the consequent damaging effects to cell survival. Devitrification times were calculated considering an initial temperature of material immersed in liquid nitrogen (−196 °C), and two temperatures of liquid nitrogen vapors within the dewar (−50 °C and −70 °C) to which the sample could be exposed for a period of time, either during storage or upon its removal. The mathematical model indicated samples could reach glass transition temperatures and undergo devitrification in 30 seconds. Results of the present study indicate storage of vitrified oocytes and embryos in the liquid nitrogen vapor phase (as opposed to completely immersed in liquid nitrogen) poses the potential risk of devitrification. Because of the reduced time-handling period before samples reach critical rubbery and devitrification values, caution should be exercised when handling samples in vapor phase.