Extra- and intra-cellular ice formation of red seabream (Pagrus major) embryos at different cooling rates

The ice crystal formation is assumed as the most lethal factor for the failure of fish embryo cryopreservation and intracellular ice formation (IIF) plays a central role in cell injury during cooling. The objectives were to observe the morphological changes of red seabream (Pagrus major) embryo during the cooling–thawing process, and to examine the effect of cryoprotectant and cooling rate on the temperatures of oil globule ice formation (T_OIF), extra-cellular ice formation (T_EIF) and intracellular ice formation (T_IIF) using cryomicroscope. After thawing, the morphological changes of embryos were observed and recorded by the video attachment and monitor under the microscope. During the cooling process, three representative phenomena were observed: oil globule gradually turned bright firstly, then the whole field of view flashed and the embryo blackened. Cooling rate affect the temperature of both extra- and intra-cellular ice formations. T_EIF and T_IIF at high cooling rate were much lower than that at low cooling rate. And the value of T_EIF − T_IIF increased from 0.45 to 11.11 °C with the increase of cooling rate from 3 to130 °C/min. Taken together, our results suggested that high cooling rate with proper cryoprotectant would be a good option for red seabream embryo cryopreservation.     

The dominance of warming rate over cooling rate in the survival of mouse oocytes subjected to a vitrification procedure

The formation of more than trace amounts of ice in cells is lethal. The two contrasting routes to avoiding it are slow equilibrium freezing and vitrification. The cryopreservation of mammalian oocytes by either method continues to be difficult, but there seems a slowly emerging consensus that vitrification procedures are somewhat better for mouse and human oocytes. The approach in these latter procedures is to load cells with high concentrations of glass-inducing solutes and cool them at rates high enough to induce the glassy state. Several devices have been developed to achieve very high cooling rates. Our study has been concerned with the relative influences of warming rate and cooling rate on the survival of mouse oocytes subjected to a vitrification procedure. Oocytes suspended in an ethylene glycol–acetamide–Ficoll–sucrose solution were cooled to −196 °C at rates ranging from 37 to 1827 °C/min between 20 and −120 °C, and for each cooling rate, warmed at rates ranging from 139 to 2950 °C/min between −70 and −35 °C. The results are unambiguous. If the samples were warmed at the highest rate, survivals were >80% over cooling rates of 187–1827 °C/min. If the samples were warmed at the lowest rate, survivals were near 0% regardless of the cooling rate. We interpret the lethality of slow warming to be a consequence of it allowing time for the growth of small intracellular ice crystals by recrystallization.     

Potential impact of rising seawater temperature on copepods due to coastal power plants in subtropical areas

The major objective of this study is to understand the upper thermal limits and potential impact of temperature elevation on copepods caused by coastal power plants. Laboratory experiments were designed to evaluate the upper incipient lethal temperature (UILT) and critical thermal maximum (CTMax) of eight coastal copepod species collected from a subtropical bay in spring and summer. The 48h-UILT of copepods acclimatized at 16.0, 20.0, 28.0 °C were 26.4-29.1, 27.3-30.1, 32.9-36.9 °C, respectively. And the CTMax of copepods acclimatized at 28.0 °C was 35.80-41.03 °C. The UILT of copepods increased significantly with rising acclimatization temperatures, but the difference values between UILT and acclimatization temperatures decreased, which indicated that the seawater temperature elevation induced the growing mortalities of copepods with increasing natural seawater temperatures from the thermal addition of power plants. The results also showed that estuarine copepods had more tolerances to the thermal stress than those from other more stable marine environments. As to the calanoid copepod species, there was a significant negative correlation between the CTMax and body length (p < 0.01). So it seemed that the copepod species with large body size were more sensitive to the thermal addition than the smaller ones. Thus, owing to the temperature increase, the copepod species diversity might reduce and the composition of copepod communities might tend to be small-sized in natural sea areas close to the coastal power plants.     

Cryopreservation of cornea: a low cooling rate improves functional survival of endothelium after freezing and thawing

AIM: To investigate the influence of low cooling rates on endothelial function and morphology of corneas frozen with propane-1,2-diol (PROH). METHODS: Rabbit corneas, mounted on support rings, were exposed to 1.4mol/l (10% v/v) PROH, seeded to initiate freezing, and cooled at 0.2 or 1 degrees C/min to -80 degrees C. Corneas were frozen immersed in liquid or suspended in air. After being held overnight in liquid nitrogen, corneas were warmed at 1 or 20 degrees C/min. After stepwise removal of the cryoprotectant, the ability of the endothelium actively to control corneal hydration was monitored during normothermic perfusion. Morphology was assessed after staining with trypan blue and alizarin red S, and by specular microscopy during perfusion. RESULTS: Functional survival was achieved only after slow cooling (0.2 degrees C/min) with the cornea immersed in the cryoprotectant medium, and rapid warming (20 degrees C/min). These conditions also gave the best morphology after freezing and thawing. CONCLUSION: Cooling rates lower than those typically applied to cornea improved functional survival of the endothelium. This result is in accord with previous observations showing the benefit of low cooling rates for cell monolayers [CryoLetters 17 (1996) 213-218].     

The cryopreservation of Chlorella

The temperature at which Chlorella 211/8h was grown determined the response to a subsequent stress of freezing to and thawing from-196°C. Cells cultured at 20°C were the most sensitive to freezing injury; at both higher and lower growth temperatures resistance to damage induced by freezing developed. At all culture temperatures examined the freezing tolerance varied with the age of culture.     

The cryopreservation of Chlorella 1. Interactions of rate of cooling,protective additive and warming rate

The cryoprotective additives glycerol and dimethylsulphoxide were found to be toxic to Chlorella cells at concentrations greater then 2.5% w/v. Polyvinylpyrrolidone, was not damaging up to a concentration of 15% w/v. Chlorella 211/7a had a recovery rate greater than 95% at all rates of cooling studied. With Chlorella 211/8h the survival was lower than 0.1% at all rates examined. The addition of dimethylsulphoxide (5% w/v) to Chlorella 211/8h increased the recovery, particularly at the faster rates of cooling; with polyvinylpyrrolidone (10% w/v) there was an optimum range of cooling rate.Cells of Chlorella 211/7a from the exponential phase of growth were found to be damaged both by a temperature reduction from 25°C to 0°C (thermal shock) and by freezing and thawing. In contrast cells from the stationary phase of growth were resistant to these stresses.Abbreviations DMSO dimethylsulphoxide - HEPES N-2-hydroxyethylpiperazine-N-2-ethansulphonic acid - PVP polyvinylpyrrolidone     

Effect of cooling rates on the cold hardiness and cryoprotectant profiles of locust eggs

To examine the relationship between cooling rate and cold hardiness in eggs of the migratory locust, Locusta migratoria, the survival rates and cryoprotectant levels of three embryonic developmental stages were measured at different cooling rates (from 0.05 to 0.8 degrees C min(-1)) in acclimated and non-acclimated eggs. Egg survival rate increased with decreasing cooling rate. The concentration of cryoprotectants (myo-inositol, trehalose, mannitol, glycerol, and sorbitol) increased in non-acclimated eggs, but varied significantly in response to different cooling rates in acclimated eggs. The acclimation process (5 degrees C for 3 days) did not increase eggs resistance to quick cooling ("plunge" cooling and 0.8 degrees C min(-1)). Earlier stage embryos were much more sensitive than later stage embryos to the same cooling rates. Time spent at subzero temperatures also had a strong influence on egg survival.     

The effect of cooling on the acetylcholine-induced current of identified <Emphasis Type="Italic">Helix pomatia</Emphasis> Br neuron

The Br neuron of the snail Helix pomatia, involved in neuronal regulation of various homeostatic and adaptive mechanisms, represents an interesting model for studying effects of temperature changes on neuronal activity of poikilotherms. The acetylcholine (ACh) induces a transient, inward dose-dependent current in the identified Br neuron. In the work presented, we analyses the effects of cooling on the ACh-induced inward current. The amplitude of ACh-induced inward current was markedly decreased after cooling and the speed of the decay of ACh response was decreased. Sensitivity to cooling of Ach-activated current on the Br neuron is mediated by a mechanism that does not involve change in the apparent receptor affinity or the cooperativity of binding.     

Discovery and development of a novel class of phenoxyacetyl amides as highly potent TRPM8 agonists for use as cooling agents

The paper presents the activity trends for a novel series of phenoxyacetyl amides as human TRPM8 receptor agonists. This series encompasses in vitro activity values ranging from the micromolar to the picomolar levels. Sensory evaluation of these molecules highlights their relevance as cooling agents for oral applications. The positive outcome of the complete evaluation of N-(1H-pyrazol-3-yl)-N-(thiophen-2-ylmethyl)-2-(p-tolyloxy)acetamide resulted in its approval for Generally Recognized As Safe (GRAS) status by the Flavor & Extract Manufacturer Association (FEMA) as FEMA 4809.     

Quantifying the cooling effect of rain events on outdoor thermal comfort in the southern coastal stations of the Caspian Sea

Pleasant outdoor thermal conditions depend on a wide range of climatic elements. The impact of rainfall events, as important climatic elements, on providing thermal comfort, has been less explored in the available literature. The work presented herein investigates the impact of Rainy Days as well as a Day Prior to (D_prior) and a Day Post rain (D_post) events on thermal conditions in the southern coastal region of the Caspian Sea. In this study, rainfall events during 1961–2017 observational period were categorized based on their intensity. Then, human thermal comfort during non-rainy (sunny) and rainy days was estimated and compared by using the radiation-driven Physiological Equivalent Temperature (PET) index, Universal Thermal Climate Index (UTCI) and Perceived Temperature (PT) index. Furthermore, difference between the average of thermal conditions in rainy days compared to a day prior and a day post rain events was calculated separately for comfort, cold and heat stress thresholds of each bioclimatic index. Finally, the correlation between the average of indices for rainy days and the frequency of rainfall events of each specific year was computed. Results suggested that overall average of studied indices for all rainy days is lower than the average for days prior and post the rain events. PET index has shown to be most impacted and reduced as a result of rain events and therefore more indicative of a cool ing effect. The observed difference in total average of PET in rainy days compared to non-rainy days were 8.30 °C, 5.86 °C and 8.85 °C for Babolsar, Rahst and Gorgan stations, respectively. Generally, the cooling effect of rain events on the temperature for a day prior rain events is higher than a day post rainfall. Finally, the trend analysis on rainy days in the studied period revealed that the average of bioclimatic indices in western stations (Babolsar and Rasht) are increasing whereas a decreasing trend was observed for Gorgan as more of an eastern station.