Peripheral nerve at extreme low temperatures 2: Pharmacologic modulation of temperature effects

Changes in temperature have profound and clinically important effects on the peripheral nerve. In a previous paper, the effects of temperature on many properties of the peripheral nerve action potential (NAP) were explored including the NAP amplitude, conduction velocity and response to paired pulse stimulation. In this paper, the effects of pharmacologic manipulations on these parameters were explored in order to further understand the mechanisms of these effects.The reduction in conduction velocity with temperature was shown to be independent of the ionic composition of the perfusate and was unaffected by potassium or sodium channel blockade. This implies that the phenomenon of reduced conduction velocities at low temperature may be related to changes in the passive properties of the axon with temperature. Blockade of sodium channels and chronic membrane depolarization produced by high perfusate potassium concentrations or high dose 4-aminopyridine impair the resistance of the nerve to hypothermia and enhance the injury to the nerve produced by cycles of cooling and rewarming. This suggests the possibility that changes in the sodium inactivation channel may be responsible for the changes in the NAP amplitude with temperature and that prolonged sodium inactivation may lead more permanent changes in excitability.     

Akt and phosphoinositide regulation and wortmannin-dependent induction of phospho-Akt in 3T3-L1 adipocytes on cold exposure followed by rewarming

We exposed 3T3-L1 adipocytes to cooling followed by rewarming and examined phospho-molecule induction, including that of phospho-Akt, and the mechanism underlying the induction in these exposed cells; the viability did not deteriorate. Cooling at 4 °C increased the phospho-Thr-308 Akt level. On subsequent rewarming at 37 °C, phospho-Ser-473 Akt expression was induced in a wortmannin (PI3K inhibitor)-dependent and anisomycin (ribotoxic agent)-independent manner without decrease in the phospho-Thr-308 Akt level, and Akt-mediated phosphorylation and PI 3,4,5-trisphosphate accumulation were induced. Our results suggest that cooling followed by rewarming induces phospho-molecules through different responses of regulatory molecules, including the target molecule, to changes in temperature.     

Numerical simulation of the effect of superparamagnetic nanoparticles on microwave rewarming of cryopreserved tissues

In this study, the microwave rewarming process of cryopreserved samples with embedded superparamagnetic (SPM) nanoparticles was numerically simulated. The Finite Element Method (FEM) was used to calculate the coupling of the electromagnetic field and the temperature field in a microwave rewarming system composed of a cylindrical resonant cavity, an antenna source, and a frozen sample phantom with temperature-dependent properties. The heat generated by the sample and the nanoparticles inside the electromagnetic field of the microwave cavity was calculated. The dielectric properties of the biological tissues were approximated using the Debye model, which is applicable at different temperatures. The numerical results showed that, during the rewarming process of the sample phantom without nanoparticles, the rewarming rate was 29.45 °C/min and the maximum temperature gradient in the sample was 3.58 °C/mm. If nanoparticles were embedded in the sample, and the cavity power was unchanged, the rewarming rate was 47.76 °C/min and the maximum temperature gradient in the sample was 1.64 °C/mm. In the presence of SPM nanoparticles, the rewarming rate and the maximum temperature gradient were able to reach 20.73 °C/min and 0.68 °C/mm at the end of the rewarming under the optimized cavity power setting, respectively. The ability to change these temperature behaviors may prevent devitrification and would greatly diminish thermal stress during the rewarming process. The results indicate that the rewarming rate and the uniformity of temperature distribution are increased by nanoparticles. This could be because nanoparticles generated heat in the sample homogeneously and the time-dependent parameters of the sample improved after nanoparticles were homogeneously embedded within it. We were thus able to estimate the positive effect of SPM nanoparticles on microwave rewarming of cryopreserved samples.     

Habituation of the metabolic and ventilatory responses to cold-water immersion in humans

An experiment was undertaken to answer long-standing questions concerning the nature of metabolic habituation in repeatedly cooled humans. It was hypothesised that repeated skin and deep-body cooling would produce such a habituation that would be specific to the magnitude of the cooling experienced, and that skin cooling alone would dampen the cold-shock but not the metabolic response to cold-water immersion. Twenty-one male participants were divided into three groups, each of which completed two experimental immersions in 12 °C water, lasting until either rectal temperature fell to 35 °C or 90 min had elapsed. Between these two immersions, the control group avoided cold exposures, whilst two experimental groups completed five additional immersions (12 °C). One experimental group repeatedly immersed for 45 min in average, resulting in deep-body (1.18 °C) and skin temperature reductions. The immersions in the second experimental group were designed to result only in skin temperature reductions, and lasted only 5 min. Only the deep-body cooling group displayed a significantly blunted metabolic response during the second experimental immersion until rectal temperature decreased by 1.18 °C, but no habituation was observed when they were cooled further. The skin cooling group showed a significant habituation in the ventilatory response during the initial 5 min of the second experimental immersion, but no alteration in the metabolic response. It is concluded that repeated falls of skin and deep-body temperature can habituate the metabolic response, which shows tissue temperature specificity. However, skin temperature cooling only will lower the cold-shock response, but appears not to elicit an alteration in the metabolic response.     

Cooling rate optimization for zebrafish sperm cryopreservation using a cryomicroscope coupled with SYBR14/PI dual staining

The Zebrafish has gained increased popularity as an aquatic model species in various research fields, and its widespread use has led to numerous mutant strains and transgenic lines. This creates the need to store these important genetic materials as frozen gametes. Sperm cryopreservation in zebrafish has been shown to yield very low post-thaw survival and many protocols suffer from great variability and poor reproducibility. The present study was intended to develop a freezing protocol that can be reliably used to cryopreserve zebrafish sperm with high post-thaw survival. In particular, our study focused on cooling protocol optimization with the aid of cryomicroscopy. Specifically, sperm suspended in 8% DMSO or 4% MeOH were first incubated with live/dead fluorescent dyes (SYBR14/PI) and then cooled at various rates from 4 °C to different intermediate stopping temperatures such as −10, −20, −30 and −80 °C before rewarming to 35 °C at the rate of 100 °C/min. %PI-positive (dead) cells were monitored throughout the cooling process and this screening yielded an optimal rate of 25 °C/min for this initial phase of freezing. We then tested the optimal cooling rate for the second phase of freezing from various intermediate stopping temperatures to −80 °C before plunging into liquid nitrogen. Our finding yielded an optimal intermediate stopping temperature of −30 °C and an optimal rate of 5 °C/min for this second phase of freezing. When we further applied this two-step cooling protocol to the conventional controlled-rate freezer, the average post-thaw motility measured by CASA was 46.8 ± 6.40% across 11 males, indicating high post-thaw survival and consistent results among different individuals. Our study indicates that cryomiscroscopy is a powerful tool to devise the optimal cooling conditions for species with sperm that are very sensitive to cryodamage.     

The use of a temperature-profiled position transducer for the study of low-temperature growth in Gramineae

A device is described for measuring linear extension of grass leaves during controlled cooling and heating of the growing region. The instrument was employed to investigate the sensitivity to temperature of the expanding third and fourth leaves of Lolium temulentum L. seedlings. Using a stepped temperature profile it was established that there was no lag in the response of growth rate to rapid changes in temperature below 16°C. If cooling was continued to the point where growth ceased (1°C) but no further, then rates of growth on rewarming were enhanced over the chilling range and reverted to the original rate at 20°C. Cooling to successively lower subzero temperatures before rewarming abolished the hysteretic enhancement, progressively raised the temperature at which growth resumed and decreased the rate of extension until, at-5.3°C, no recovery occurred. The temperature sensitivity of growth, measured as Q₁₀, was essentially constant when cooling from 20°C to 5°C, with 5°C-grown leaf tissue exhibiting a higher mean Q₁₀ than tissue developed at 20°C. The possible physiological significance of these data is discussed.Abbreviations LVDT linear variable displacement transformer - Pe, Fx temperatures at which growth ceases during cooling and resumes during rewarming     

Cryopreservation of carotid artery segments via vitrification subject to marginal thermal conditions: correlation of freezing visualization with functional recovery

Cryopreservation is a well-established technique for long-term storage of viable cells and tissues. However, in recent years, application of established cryobiological principles to the preservation of multicellular tissues and organs has demanded considerable attention to ways of circumventing the deleterious effects of ice and thermal stresses in bulky tissues. As part of a multidisciplinary research program designed to study the interactions of thermo-physical events with tissue preservation, we report here on the implementation of a slow cooling (3 °C/min) and slow warming (62 °C/min) regimen towards scale-up of vitreous preservation of large tissue samples. Specifically, the correlation of thermo-physical events during vitrification of carotid artery segments with function recovery is reported using marginal thermal conditions for achieving vitrification in bulky samples. Moreover, the outcome is compared with a similar study reported previously using a 3-fold higher rate of rewarming (186 ± 13 °C/min). Tissue vitrification using an 8.4 M cryoprotectant cocktail solution (VS55) was achieved in 1 ml samples by imposing a low (2.6 ± 0.1 °C/min) cooling rate, between −40 and −100 °C, and a low rewarming rate (62 ± 4 °C/min) between −100 and −40 °C. Following cryoprotectant removal, the artery segments were cut into 3-4 mm rings for function testing on a contractility apparatus by measuring isometric responses to four agonist and antagonists (norepinephrine, phenylepinephrine, calcium ionophore and sodium nitroprusside). In addition, non-specific metabolic function of the vessel rings was determined using the REDOX indicator alamarBlue. Contractile function, normalized to untreated control samples, in response to the agonists norepinephrine and phenylepinephrine was significantly better in the slowly rewarmed group of carotid segments (74 ± 9% and 62 ± 11%, respectively) than for the more rapidly warmed group 31 ± 7% and 45 ± 15%, respectively). However, EC₅₀ sensitivities were not significantly different between the groups. Thermo-physical events such as ice formation and fractures were monitored throughout the cooling and warming phases using cryomacroscopy with the aid of a purpose-built borescope device. This technique allowed a direct observation of the visual impact of ice formation on specific zones along the blood vessel segment where, in most cases, no ice formation or fractures were observed in the vicinity of the artery segments. However, in specific instances when some ice crystallization was observed to impact the artery segment, the subsequent testing of function revealed a total loss of contractility. The successful vitrification of blood vessel segments using marginal conditions of slow cooling and rewarming, provide essential information for the development of scale-up protocols that is necessary when clinically relevant size samples need to be cryopreserved in an essentially ice-free state. This information can further be integrated into computer simulations of heat transfer and thermo-mechanical stress, where the slowest cooling rate anywhere in the simulated domain must exceed the critical values identified in the current study.     

The use of cryomicroscopy in guppy sperm freezing

The present study employed cryomicroscopy to derive an optimal sperm freezing protocol for guppy (Poecilia reticulata) sperm. Evaluation criteria during the freezing-thawing process were assessed for nucleation temperature (Tn), temperature when more than 50% of sperm display bending mid-piece (Tb), temperature when more than 80% of sperm stop moving (Tm), thawing temperature (Tt), and post-thaw motility. We compared four different cryoprotectants: 5% N-dimethyl formamide (DMF), 6% methanol (MEOH), 10% dimethyl sulfoxide (DMSO), and 14% glycerol, as well as glycerol at different concentrations of 7-50%; cooling and rewarming rates ranged from 5 to 100 °C/min. The protocol that yielded the highest post-thaw motility was samples suspended in 14% glycerol, cooled at 25 °C/min, and thawed at 100 °C/min, which was in complete agreement with our previous findings derived from a controlled-rate freezer. In addition, Tb and Tm were found to be negatively correlated with post-thaw motility, suggesting their possible role in predicting freezing success. The present study for the first time demonstrated the usefulness of cryomicroscopy in deriving an optimal sperm freezing protocol for aquatic species.     

Rapid thermal responses and thermal tolerance in adult codling moth Cydia pomonella (Lepidoptera: Tortricidae)

In order to preserve key activities or improve survival, insects facing variable and unfavourable thermal environments may employ physiological adjustments on a daily basis. Here, we investigate the survival of laboratory-reared adult Cydia pomonella at high or low temperatures and their responses to pre-treatments at sub-lethal temperatures over short time-scales. We also determined critical thermal limits (CTLs) of activity of C. pomonella and the effect of different rates of cooling or heating on CTLs to complement the survival assays. Temperature and duration of exposure significantly affected adult C. pomonella survival with more extreme temperatures and/or longer durations proving to be more lethal. Lethal temperatures, explored between −20 °C to −5 °C and 32 °C to 47 °C over 0.5, 1, 2, 3 and 4 h exposures, for 50% of the population of adult C. pomonella were −12 °C for 2 h and 44 °C for 2 h. Investigation of rapid thermal responses (i.e. hardening) found limited low temperature responses but more pronounced high temperature responses. For example, C. pomonella pre-treated for 2 h at 5 °C improved survival at −9 °C for 2 h from 50% to 90% (p < 0.001). At high temperatures, pre-treatment at 37 °C for 1 h markedly improved survival at 43 °C for 2 h from 20% to 90% (p < 0.0001). We also examined cross-tolerance of thermal stressors. Here, low temperature pre-treatments did not improve high temperature survival, while high temperature pre-treatment (37 °C for 1 h) significantly improved low temperature survival (−9 °C for 2 h). Inducible cross-tolerance implicates a heat shock protein response. Critical thermal minima (CTmin) were not significantly affected by cooling at rates of 0.06, 0.12 and 0.25 °C min⁻¹ (CTmin range: 0.3-1.3 °C). By contrast, critical thermal maxima (CTmax) were significantly affected by heating at these rates and ranged from 42.5 to 44.9 °C. In sum, these results suggest pronounced plasticity of acute high temperature tolerance in adult C. pomonella, but limited acute low temperature responses. We discuss these results in the context of local agroecosystem microclimate recordings. These responses are significant to pest control programmes presently underway and have implications for understanding the evolution of thermal tolerance in these and other insects.     

Rewarming rates of two large hibernators: Comparison of a monotreme and a eutherian

We measured body temperatures in two large hibernating mammals, the eutherian alpine marmot (Marmota marmota) and the egg-laying echidna (Tachyglossus aculeatus) from unrestrained animals in their natural environment. In both species hibernation is broken every 13 days on average by rewarming to euthermic temperatures. We found that the time course of a rewarming could be closely fitted with a sigmoid curve, allowing calculation of peak rewarming rate and corresponding body temperature. Maximum rewarming rates were twice as high in marmots as in echidnas (12.1±1.3 °C h⁻¹, n=10 cf. 6.2±1.2 °C h⁻¹, n=10). Peak rewarming rates were positively correlated with body temperature in echidnas, but negatively correlated in marmots.     

Thermal tolerance of European Sea Bass (Dicentrarchus labrax) juveniles acclimated to three temperature levels

This study was carried out to determine upper (CTMax) and lower (CTMin) thermal tolerance, acclimation response ratio (ARR) and thermal tolerance polygon of the European sea bass inhabiting the Iskenderun Bay, the most southeasterly part of the Mediterranean Sea, at three acclimation temperatures (15, 20, 25 °C). Acclimation temperature significantly affected the CTMin and CTMax values of the fish. At 0.3 °C min⁻¹ cooling or heating rate, CTMin ranged from 4.10 to 6.77 °C and CTMax ranged from 33.23 to 35.95 °C in three acclimation temperatures from 15 to 25 °C. Thermal tolerance polygon for the juveniles at the tested acclimation temperatures was calculated to be 296.14 °C². In general, the current data show that our sea bass population possesses acclimation response ratio (ARR) values (0.25-0.27) similar to some tropical species. The cold tolerance values attained for this species ranged from 4.10 to 6.77 °C, suggesting that cold winter temperatures may not pose danger during the culture of European sea bass in deep ponds or high water exchange rate systems. Upper thermal tolerance is more of a problem in the southern part of the Mediterranean as maximum water temperature in ponds may sometimes exceed 33-34 °C, during which underground cool-water should be used to lower ambient water temperature in the mid-summer. For successful culture of sea bass in ponds, temperature should be maintained around 25 °C throughout the year and this can be managed under greenhousing systems using underground well-waters, commonly available in the region.     

Myocardial mechanical dysfunction and calcium overload following rewarming from experimental hypothermia in vivo

Rewarming patients from accidental hypothermia are regularly complicated with cardiovascular instability ranging from minor depression of cardiac output to fatal circulatory collapse also termed “rewarming shock”. Since altered Ca²⁺ handling may play a role in hypothermia-induced heart failure, we studied changes in Ca²⁺ homeostasis in in situ hearts following hypothermia and rewarming. A rat model designed for studies of the intact heart in a non-arrested state during hypothermia and rewarming was used. Rats were core cooled to 15 °C, maintained at 15 °C for 4 h and thereafter rewarmed. As time-matched controls, one group of animals was kept at 37 °C for 5 h. Total intracellular myocardial Ca²⁺ content ([Ca²⁺]_i) was measured using ⁴⁵Ca²⁺. Following rewarming we found a significant reduction of stroke volume and cardiac output compared to prehypothermic control values as well as to time-matched controls. Likewise, we found that hypothermia and rewarming resulted in a more than six-fold increase in [Ca²⁺]_i to 3.01 ± 0.43 μmol/g dry weight compared to 0.44 ± 0.05 μmol/g dry weight in normothemia control. These findings indicate that hypothermia-induced alterations in the Ca²⁺-handling result in Ca²⁺ overload during hypothermia, which may contribute to myocardial failure during and after rewarming.     

In vitro characteristics of frozen-thawed, sex-sorted bull sperm after refreezing or incubation at 15 or 37 °C

The objective was to determine the in vitro characteristics of frozen-thawed dairy bull sperm after sex-sorting and refreezing and thawing (0, 2, and 4 h post-thaw; 37 °C) or post-sort incubation at 15 or 37 °C for 30 and 24 h, respectively. These sperm were compared with nonsorted frozen-thawed sperm (control) and with nonsorted sperm undergoing two cryopreservation procedures (FF; 0, 2, and 4 h). Frozen-thawed sex-sorted (FS) sperm maintained at 15 or 37 °C had higher (P < 0.001) progressive motility (PM), velocity, mitochondrial function, viability, and acrosome integrity than that of control sperm but similar total motility at 0 and 2 h of incubation. Frozen-thawed sex-sorted sperm incubated at 15 °C maintained high levels of motility (66.5 ± 1.6%) and viability/acrosome integrity (64.9 ± 1.2%) at 24 h incubation and, after rewarming and further 6 h incubation at 37 °C, acceptable levels of motility (35.8 ± 1.6%) and viability/acrosome integrity (51.2 ± 1.2%) were maintained. Frozen-thawed sex-sorted sperm maintained at 37 °C had lower levels of motility, integrity, mitochondrial respiration, and velocity from 4 h of incubation onward than that of those incubated at 15 °C. However, when frozen-thawed sex-sorted sperm were refrozen (FSF), motility and velocity were depressed at all hours compared with levels exhibited by control sperm, but membrane viability/acrosome integrity and mitochondrial respiration were similar at 0 and 2 h post-thaw. Acrosome integrity of sperm in all groups undergoing sorting was exceptionally high at 0 h (≥90%), even after re-cryopreservation and 4 h of incubation (77.5 ± 1.3%). Double frozen-thawed nonsorted sperm (FF) had similar motility to FSF sperm at 0 and 2 h post-thaw but at all time points had the lowest (P < 0.001) levels of acrosome intact/viable sperm and mitochondrial respiration and the lowest velocity at 0 h. In conclusion, whereas sex-sorting improved the functionality of frozen-thawed sperm, refreezing depressed motility, viability, and velocity but not acrosome integrity after extended incubation compared with that of control sperm. Furthermore, frozen-thawed, sex-sorted sperm may be stored for transport at 15 °C for up 24 h without detrimental effects on in vitro sperm characteristics.     

Force variability depends on core and muscle temperature

The aim of this study was to investigate if voluntary activation and force variability during maximal voluntary contraction (MVC) depends more on muscle (local) or body (core) temperature. Ten volunteers performed a 2-min MVC of the knee extensors under the control (CON) conditions (ambient temperature (21 °C), relative humidity (30%), and air velocity (∼0.1 m/s)) as well as after heating (HT) and cooling (CL) of the lower body. During water manipulation procedure lower body was immersed up to the waist in a water bath at ∼44 °C for 45 min for HT experiment, and ∼15 °C for 30 min for CL experiment. Peak torque, torque variability, muscle voluntary activation and half-relaxation time were assessed during the exercise. HT increased muscle (2.8±0.2 °C) and rectal (1.9±0.1 °C) temperatures while CL lowered muscle (2.2±0.2 °C) temperature, but did not affect rectal temperature. During 2-min MVC, peak torque decreased (P<0.05; SP>90%) and to a lower level in HT compared to CON and CL experiments (52.6±2.3% versus 69.0±2.3% and 65.6±1.9% MVC, respectively, P<0.05; SP>90%). Torque variability increased significantly during exercise and was significantly larger in HT and lower in CL compared to CON experiment. Voluntary activation of exercising muscle was more depressed in HT (i.e. greater central fatigue) and the smallest effect was found in CL compared to CON. In conclusion increased core and muscle temperature impairs voluntary activation and increases force variability of the exercising muscles while a local muscle cooling decrease force variability but has a small effect on central fatigue.     

Combined effects of temperature and salinity on critical thermal minima of pacific white shrimp Litopenaeus vannamei (Crustacea: Penaeidae)

Critical thermal minima (CTMin) were determined for the Pacific white shrimp Litopenaeus vannamei juveniles from four different acclimation temperatures (15, 20, 25, and 30 °C) and salinities (10‰, 20‰, 30‰, and 40‰). The lowest and highest CTMin of shrimp ranged between 7.2 °C at 15 °C/30‰ and 11.44 °C at 30 °C/20‰ at the cooling rate of 1 °C h⁻¹. Acclimation temperature and salinity, as well as the interaction of both parameters, had significant effects on the CTMin values of L. vannamei (P<0.01). Yet, the results showed a much more profound effect of temperature on low thermal tolerance of juveniles. Only 40‰ salinity had an influence on the CTMin values (P<0.01). As the acclimation temperature was lowered from 30 to 15 °C thermal tolerance of the shrimp significantly increased by 3.25–4.14 °C. The acclimation response ratio (ARR) of the Pacific white shrimp exposed to different combinations of salinity and temperature ranged between 0.25 and 0.27. When this species is farmed in sub-tropical regions, its pond water temperature in the over-wintering facilities (regardless of the water salinity level) must never fall below 12 °C throughout the cold season to prevent mortalities.     

Determination of the water permeability (Lp) of mouse oocytes at −25 °C and its activation energy at subzero temperatures

Typically, subzero permeability measurements are experimentally difficult and infrequently reported. Here we report an approach we have applied to mouse oocytes. Interrupted cooling involves rapidly cooling oocytes (50 °C/min) to an intermediate temperature above the intracellular nucleation zone, holding them for up to 40 min while they dehydrate, and then rapidly cooling them to −70 °C or below. If the intermediate holding temperature and holding time are well chosen, high post thaw survival of the oocytes is possible because the freezable water is removed during the hold. The length of time required for the exit of the freezable water allows the water permeability at that temperature to be determined. These experiments used 1.5 M ethylene glycol in PBS and included a transient hold of 2 min for equilibration at −10 °C, just below the extracellar ice formation temperature. We obtain an Lp = 1.8 × 10⁻³ μm min⁻¹ atm⁻¹ at −25 °C based on a hold time of 30 min yielding 80% survival and the premise that most of the freezable water is removed during the 30 min hold. If we assume that the water permeability is a continuous function of temperature and that its Ea changes at 0 °C, we obtain a subzero Ea of 21 kcal/mol; higher than the suprazero value of 14 kcal/mol. A number of assumptions are required for these water loss calculations and the resulting value of Lp can vary by up to a factor of 2, depending on the choices make.     

Cold tolerance of first-instar nymphs of the Australian plague locust, Chortoicetes terminifera

The cold tolerance of first-instar nymphs of the Australian plague locust, Chortoicetes terminifera, was examined using measures of total body water content, supercooling point and mortality for a range of sub-zero temperature exposure regimes. The supercooling points for starved and fed nymphs were −13.1 ± 0.9 and −12.6 ± 1.6 °C, and freezing caused complete mortality. Above these temperatures, nymphs were cold tolerant to different degrees based on whether they were starved or given access to food and water for 24 h prior to exposure. The rate of cooling also had a significant effect on mortality. Very rapid cooling to −7 °C caused 84 and 87% mortality for starved and fed nymphs respectively, but this significantly decreased for starved nymphs if temperature declined by more ecologically realistic rates of 0.5 and 0.1 °C min⁻¹. These results are indicative of a rapid cold hardening response and are discussed in terms of the likely effects of cold nights and frost on first-instar nymphal survival in the field.     

Post-cryopreservation viability of the benthic freshwater diatom Planothidium frequentissimum depends on light levels

Over recent years, several planktonic and benthic freshwater diatom taxa have been established as laboratory model strains. In common with most freshwater diatoms the pennate diatom Planothidium frequentissimum suffers irreversible cell shrinkage on prolonged maintenance by serial transfers, without induction of the sexual cycle. Therefore, alternative strategies are required for the long-term maintenance of this strain. Conventional colligative cryopreservation approaches have previously proven unsuccessful with no regrowth. However, in this study using 5% dimethyl sulfoxide (Me₂SO), controlled cooling at 1 °C min⁻¹, automated ice seeding and cooling to −40 °C with a final plunge into liquid nitrogen, viability levels were enhanced from 0.3 ± 0.4% to 80 ± 3%, by incorporating a 48 h dark-recovery phase after rewarming. Omission, or reduction, of this recovery step resulted in obvious cell damage with photo-bleaching of pigments, indicative of oxidative-stress induced cell damage, with subsequent deterioration of cellular architecture.     

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.     

Intracellular ice formation in yeast cells vs. cooling rate: Predictions from modeling vs. experimental observations by differential scanning calorimetry

To survive freezing, cells must not undergo internal ice formation during cooling. One vital factor is the cooling rate. The faster cells are cooled, the more their contents supercool, and at some subzero temperature that supercooled cytoplasm will freeze. The question is at what temperature? The relation between cooling rate and cell supercooling can be computed. Two important parameters are the water permeability (Lp) and its temperature dependence. To avoid intracellular ice formation (IIF), the supercooling must be eliminated by dehydration before the cell cools to its ice nucleation temperature. With an observed nucleation temperature of −25 °C, the modeling predicts that IIF should not occur in yeast cooled at <20 °C/min and it should occur with near certainty in cells cooled at ?30 °C/min. Experiments with differential scanning calorimetry (DSC) confirmed these predictions closely. The premise with the DSC is that if there is no IIF, one should see only a single exotherm representing the freezing of the external water. If IIF occurs, one should see a second, lower temperature exotherm. A further test of whether this second exotherm is IIF is whether it disappears on repeated freezing. IIF disrupts the plasma membrane; consequently, in a subsequent freeze cycle, the cell can no longer supercool and will not exhibit a second exotherm. This proved to be the case at cooling rates >20 °C/min.