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Differential thermal analysis (DTA) has indicated that xylem ray parenchyma cells (XRPCs) of hardwood species adapt to freezing of apoplastic water either by deep supercooling or by extracellular freezing, depending upon the species. DTA studies indicated that moderately cold hardy hardwood species exhibiting deep supercooling in the XRPCs were limited in latitudinal distribution within the -40 degrees C isotherm, while very hardy hardwood species exhibiting extracellular freezing could distribute in colder areas beyond the -40 degrees C isotherm. Predictions based on the results of DTA, however, indicate that XRPCs exhibiting extracellular freezing may appear not only in very hardy woody species native to cold areas beyond the -40 degrees C isotherm but also in less hardy hardwood species native to tropical and subtropical zones as well as in a small number of moderately hardy hardwood species native to warm temperate zones. Cryo-scanning electron microscopic (cryo-SEM) studies on the freezing behavior of XRPCs have revealed some errors in DTA. These errors are originated mainly due to the overlap between exotherms produced by freezing of water in apoplastic spaces (high temperature exotherms, HTEs) and exotherms produced by freezing of intracellular water of XRPCs by breakdown of deep supercooling (low temperature exotherms, LTEs), as well as to the shortage of LTEs produced by intracellular freezing of XRPCs. In addition, DTA results are significantly affected by cooling rates employed. Further, cryo-SEM observations, which revealed the true freezing behavior of XRPCs, changed the previous knowledge of freezing behavior of XRPCs that had been obtained by freeze-substitution and transmission electron microscopic studies. Cryo-SEM results, in association with results obtained from DTA that were reconfirmed or changed by observation using a cryo-SEM, revealed a clear tendency of the freezing behavior of XRPCs in hardwood species to change with changes in the temperature in the growing conditions, including both latitudinal and seasonal temperature changes. In latitudinal temperature changes, XRPCs in less hardy hardwood species native to tropical and subtropical zones exhibited deep supercooling to -10 degrees C, XRPCs in moderately hardy hardwood species native to temperate zones exhibited a gradual increase in the supercooling ability to -40 degrees C from warm toward cool temperate zones, and XRPCs in very hardy hardwood species native to boreal forests exhibited extracellular freezing via an intermediate form of freezing behavior between deep supercooling and extracellular freezing. In seasonal temperature changes, XRPCs in hardwood species native to temperate zones changed their supercooling ability from a relatively low degree in summer to a high degree in winter. XRPCs in hardwood species native to boreal forests changed their freezing behavior from deep supercooling to -10 degrees C in summer to extracellular freezing in winter. These results indicate that the freezing behavior of XRPCs in hardwood species tends to shift gradually from supercooling of -10 degrees C, to a gradual increase in the deep supercooling ability to -40 degrees C or less, and finally to extracellular freezing as a result of cold acclimation in response to both latitudinal and seasonal temperature changes. It is thought that these temperature-dependent changes in the freezing behavior of XRPCs in hardwood species are mainly controlled by changes in cell wall properties, although no distinct changes were detected by electron microscopic observations in cell wall organization between hardwood species or between seasons. Evidence of temperature-dependent changes in the freezing behavior of XRPCs in hardwood species provided by the results of studies using a cryo-SEM has indicated the need for further investigation to clarify cold acclimation-induced cell wall changes at the sub-electron microscopic level in order to understand the mechanisms of freezing adaptation.
Given the increasing importance of reducing development time for new pharmaceutical products, formulation and process development scientists must continually look for ways to "work smarter, not harder." Within the product development arena, this means reducing the amount of trial and error empiricism in arriving at a formulation and identification of processing conditions which will result in a quality final dosage form. Characterization of the freezing behavior of the intended formulation is necessary for developing processing conditions which will result in the shortest drying time while maintaining all critical quality attributes of the freeze-dried product. Analysis of frozen systems was discussed in detail, particularly with respect to the glass transition as the physical event underlying collapse during freeze-drying, eutectic mixture formation, and crystallization events upon warming of frozen systems. Experiments to determine how freezing and freeze-drying behavior is affected by changes in the composition of the formulation are often useful in establishing the "robustness" of a formulation. It is not uncommon for seemingly subtle changes in composition of the formulation, such as a change in formulation pH, buffer salt, drug concentration, or an additional excipient, to result in striking differences in freezing and freeze-drying behavior. With regard to selecting a formulation, it is wise to keep the formulation as simple as possible. If a buffer is needed, a minimum concentration should be used. The same principle applies to added salts: If used at all, the concentration should be kept to a minimum. For many proteins a combination of an amorphous excipient, such as a disaccharide, and a crystallizing excipient, such as glycine, will result in a suitable combination of chemical stability and physical stability of the freeze-dried solid. Concepts of heat and mass transfer are valuable in rational design of processing conditions. Heat transfer by conduction--the dominant mechanism of heat transfer in freeze-drying--is inefficient at the pressures used in freeze-drying. Steps should be taken to improve the thermal contact between the product and the shelf of the freeze dryer, such as eliminating metal trays from the drying process. Quantitation of the heat transfer coefficient for the geometry used is a useful way of assessing the impact of changes in the system such as elimination of product trays and changes in the vial. Because heat transfer by conduction through the vapor increases with increasing pressure, the commonly held point of view that "the lower the pressure, the better" is not true with respect to process efficiency. The optimum pressure for a given product is a function of the temperature at which freeze-drying is carried out, and lower pressures are needed at low product temperatures. The controlling resistance to mass transfer is almost always the resistance of the partially dried solids above the submination interface. This resistance can be minimized by avoiding fill volumes of more than about half the volume of the container. The development scientist should also recognize that very high concentrations of solute may not be appropriate for optimum freeze-drying, particularly if the resistance of the dried product layer increases sharply with concentration. Although the last 10 years has seen the publication of a significant body of literature of great value in allowing development scientists and engineers to "work smarter," there is still much work needed in both the science and the technology of freeze-drying. Scientific development is needed for improving analytical methodology for characterization of frozen systems and freeze-dried solids. A better understanding of the relationship between molecular mobility and reactivity is needed to allow accurate prediction of product stability at the intended storage temperature based on accelerated stability at higher temperatures. This requires that the temperature dependence of glass transition-associated mobility, particularly at temperatures below the glass transition, be studied in greater depth. The relevance of the concept of strong and fragile glasses to frozen systems and freeze-dried solids has only begun to be explored. The list of pharmaceutically acceptable protective solutes is very short, and more imagination--and work--is needed in order to develop pharmaceutically acceptable alternative stabilizers. There is a need for technology development in process monitoring, particularly in developing a way to measure the status of the product during freezing and freeze-drying without placing temperature measurement probes in individual vials of product. The current practice of placing thermocouples in vials is uncertain with respect to reliability of the data, inconsistent with elimination of personnel in close proximity to open vials of product in an aseptic environment, and incompatible with technology for automatic material handling in freeze-drying. In addition, a method for controlling the degree of supercooling during freezing would allow better control of freezing rate and would, in many cases, result in more consistent product quality.
OBJECTIVE: To evaluate the influence of cryopreservation medium and freezing-thawing techniques on human sperm motility and morphology. STUDY DESIGN: 63 semen samples were obtained from 39 donors to the artificial insemination programme. Possible effects of the sperm dilution with cryomedium on the motility were examined 10 min after exposure of 24 high initial quality semen samples to TEST-yolk ¿zwitterion-citrate-egg yolk extender containing TES [N-Tris (hydroxymethyl) methylaminoethane sulfonic acid] and Tris [(hydroxymethyl) aminomethane]¿ and human sperm preservation medium (HSPM). Post-thaw sperm motility from 24 frozen semen samples was examined comparing the cryoprotective efficacy of TEST-yolk and HSPM following different freezing techniques (vapour freezing, fast programmable freezing and slow programmable freezing). The relationship of sperm morphology to the effects of freezing was investigated on 39 semen samples following different freezing techniques. Post-thaw sperm motility from 39 frozen semen samples was compared among three groups divided according to the percentage of morphologically normal cells (<40, 40-50 and >50%) in fresh semen. RESULTS: Exposure of spermatozoa to cryomedia for 10 min at room temperature significantly reduced motility in TEST-yolk treatment group for 9% and in HSPM treatment group for 18% (P<0.01). The recovery of motile sperms (mean+/-standard deviation) was 49+/-15.7, 43+/-15.2 and 52+/-16.8% when TEST-yolk was used and 34+/-17.8, 32+/-18.2 and 50+/-13.6% when HSPM was used as a cryopreservative following vapour freezing, and fast and slow programmable freezing, respectively. Following vapour freezing and also following fast programmable freezing, the recovery of motile sperm was significantly higher (P<0.05) after addition of TEST-yolk medium than after addition of HSPM. Post-thaw motility of the sperm cryopreserved in HSPM showed significant differences (P<0.05) after three different freezing techniques. The recovery of motile sperms was 57+/-26.4, 38+/-8.6 and 38+/-17.3% in groups with >50, 40-50 and <40% morphologically normal cells, respectively. The percentage of morphologically normal spermatozoa was reduced 8% after vapour freezing and 6 and 3% after fast and slow programmable freezing, respectively. The results were statistically analysed using SAS/STAT software. CONCLUSIONS: Slow programmable freezing was superior to vapour freezing and fast programmable freezing as a method for sperm cryopreservation. However, none of these methods of freezing had discernible effects on sperm morphology. Motility of spermatozoa decreased due to the exposure of semen to cryomedium. TEST-yolk was a superior cryomedium to HSPM. Fresh semen with more than 50% of morphologically normal cells showed the best recovery of motile cells after freezing and thawing.
Patients with Parkinson's disease (PD) often experience freezing of gait, a debilitating phenomenon during which the subject suddenly becomes unable to start walking or to continue to move forward. Little is known about the gait of those subjects with PD who experience freezing of gait or the pathophysiology of freezing. One possibility is that freezing of gait is a truly paroxysmal phenomenon and that the usual walking pattern of subjects who experience freezing of gait is not different than that of other patients with PD who do not experience these transient episodes of freezing of gait. On the other hand, a recent study noted gait changes just prior to freezing and concluded that dyscontrol of the cadence of walking contributes to freezing. To address this question, we compared the gait of PD subjects with freezing of gait to PD subjects without freezing of gait. Given the potential importance of the dyscontrol of the cadence of walking in freezing, we focused on two aspects of gait dynamics: the average stride time (the inverse of cadence, a measure of the walking pace or rate) and the variability of the stride time (a measure of "dyscontrol," arrhythmicity and unsteadiness). We found that although the average stride time was similar in subjects with and without freezing, stride-to-stride variability was markedly increased among PD subjects with freezing of gait compared to those without freezing of gait, both while "on" (P<0.020) and "off" (P<0.002) anti-parkinsonian medications. Further, we found that increased gait variability was not related to other measures of motor control (while off medications) and levodopa apparently reduced gait variability, both in subjects with and without freezing. These results suggest that a paradigm shift should take place in our view of freezing of gait. PD subjects with freezing of gait have a continuous gait disturbance: the ability to regulate the stride-to-stride variations in gait timing and maintain a stable walking rhythm is markedly impaired in subjects with freezing of gait. In addition, these findings suggest that the inability to control cadence might play an important role in this debilitating phenomenon and highlight the key role of dopamine-mediated pathways in the stride-to-stride regulation of walking.
The freeze denaturation of model proteins, LDH, ADH, and catalase, was investigated in absence of cryoprotectants using a microcryostage under well-controlled freezing and thawing rates. Most of the experimental data were obtained from a study using a dilute solution with an enzyme concentration of 0.025 g/l. The dependence of activity recovery of proteins on the freezing and thawing rates showed a reciprocal and independent effect, that is, slow freezing (at a freezing rate about 1 degrees C/min) and fast thawing (at a thawing rate >10 degrees C/min) produced higher activity recovery, whereas fast freezing with slow thawing resulted in more severe damage to proteins. With minimizing the freezing concentration and pH change of buffer solution by using a potassium phosphate buffer, this phenomenon could be ascribed to surface-induced denaturation during freezing and thawing process. Upon the fast freezing (e.g., when the freezing rate >20 degrees C/min), small ice crystals and a relatively large surface area of ice-liquid interface are formed, which increases the exposure of protein molecules to the ice-liquid interface and hence increases the damage to the proteins. During thawing, additional damage to proteins is caused by recrystallization process. Recrystallization exerts additional interfacial tension or shear on the entrapped proteins and hence causes additional damage to the latter. When buffer solutes participated during freezing, the activity recovery of proteins after freezing and thawing decreased due to the change of buffer solution pH during freezing. However, the patterns of the dependence on freezing and thawing rates of activity recovery did not change except for that at extreme low freezing rates (<0.5 degrees C/min). The results exhibited that the freezing damage of protein in aqueous solutions could be reduced by changing the buffer type and composition and by optimizing the freezing-thawing protocol.
The ability of cells to survive freezing and thawing is expected to depend on the physiological conditions experienced prior to freezing. We examined factors affecting yeast cell survival during freeze-thaw stress, including those associated with growth phase, requirement for mitochondrial functions, and prior stress treatment(s), and the role played by relevant signal transduction pathways. The yeast Saccharomyces cerevisiae was frozen at -20 degrees C for 2 h (cooling rate, less than 4 degrees C min-1) and thawed on ice for 40 min. Supercooling occurred without reducing cell survival and was followed by freezing. Loss of viability was proportional to the freezing duration, indicating that freezing is the main determinant of freeze-thaw damage. Regardless of the carbon source used, the wild-type strain and an isogenic petite mutant ([rho 0]) showed the same pattern of freeze-thaw tolerance throughout growth, i.e., high resistance during lag phase and low resistance during log phase, indicating that the response to freeze-thaw stress is growth phase specific and not controlled by glucose repression. In addition, respiratory ability and functional mitochondria are necessary to confer full resistance to freeze-thaw stress. Both nitrogen and carbon source starvation led to freeze-thaw tolerance. The use of strains affected in the RAS-cyclic AMP (RAS-cAMP) pathway or supplementation of an rca1 mutant (defective in the cAMP phosphodiesterase gene) with cAMP showed that the freeze-thaw response of yeast is under the control of the RAS-cAMP pathway. Yeast did not adapt to freeze-thaw stress following repeated freeze-thaw treatment with or without a recovery period between freeze-thaw cycles, nor could it adapt following pretreatment by cold shock. However, freeze-thaw tolerance of yeast cells was induced during fermentative and respiratory growth by pretreatment with H2O2, cycloheximide, mild heat shock, or NaCl, indicating that cross protection between freeze-thaw stress and a limited number of other types of stress exists.
Increases in liver glycogen phosphorylase activity, along with inhibition of glycogen synthetase and phosphofructokinase-1, are associated with elevated cryoprotectant (glucose) levels during freezing in some freeze-tolerant anurans. In contrast, freeze-tolerant chorus frogs, Pseudacris triseriata, accumulate glucose during freezing but exhibit no increase in phosphorylase activity following 24-h freezing bouts. In the present study, chorus frogs were frozen for 5- and 30-min and 2- and 24-h durations. After freezing, glucose, glycogen, and glycogen phosphorylase and synthetase activities were measured in leg muscle and liver to determine if enzyme activities varied over shorter freezing durations, along with glucose accumulation. Liver and muscle glucose levels rose significantly (5-12-fold) during freezing. Glycogen showed no significant temporal variation in liver, but in muscle, glycogen was significantly elevated after 24 h of freezing relative to 5 and 30 min-frozen treatments. Hepatic phosphorylase a and total phosphorylase activities, as well as the percent of the enzyme in the active form, showed no significant temporal variation following freezing. Muscle phosphorylase a activity and percent active form increased significantly after 24 h of freezing, suggesting some enhancement of enzyme function following freezing in muscle. However, the significance of this enhanced activity is uncertain because of the concurrent increase in muscle glycogen with freezing. Neither glucose 6-phosphate independent (I) nor total glycogen synthetase activities were reduced in liver or muscle during freezing. Thus, chorus frogs displayed typical cryoprotectant accumulation compared with other freeze-tolerant anurans, but freezing did not significantly alter activities of hepatic enzymes associated with glycogen metabolism.
BACKGROUND: It has become routine practice in many centers to use two successive freeze-thaw cycles in the treatment of the common types of basal cell carcinoma. Because of the potential morbidity caused by this, we have investigated the cure rate achieved with one freeze-thaw cycle compared with that achieved with two freeze-thaw cycles in the treatment of facial basal cell carcinomas of a uniform type and clinically in the best prognostic group. Superficial truncal basal cell carcinomas are reported to respond to less aggressive cryosurgery, and we have investigated the cure rate achieved with one freeze-thaw cycle. OBJECTIVE: To compare the efficacy of one freeze-thaw cycle versus two freeze-thaw cycles in the treatment of facial basal cell carcinomas. Second, to investigate the efficacy of one freeze-thaw cycle in the treatment of superficial truncal basal cell carcinomas. This was investigated in a prospective randomized post-treatment follow-up study. METHODS: Over the past 7 years, we have treated 84 facial basal cell carcinomas with either a single 30-second freeze-thaw cycle or a double 30-second freeze-thaw cycle. Patients were allocated randomly into one of the two treatment schedules, and the cure rates achieved were compared. Second, 29 superficial truncal basal cell carcinomas were treated with a single 30-second freeze-thaw cycle. Patients were followed up to assess response to therapy. RESULTS: A 95.3% cure rate was achieved in the treatment of facial basal cell carcinomas with a double freeze-thaw cycle. This compared with a cure rate of only 79.4% when facial lesions were treated with a single freeze-thaw cycle. Treatment of superficial truncal basal cell carcinomas with a single freeze-thaw cycle achieved a cure rate of 95.5%. CONCLUSION: We recommend that, in order to achieve high cure rates that are equivalent to many reports of formal excision or radiotherapy, facial basal cell carcinomas require a double freeze-thaw cycle with liquid nitrogen. One freeze-thaw cycle to truncal basal cell carcinomas achieves high cure rates, equal to that achieved with a double freeze-thaw cycle to facial basal cell carcinomas.
Freezing or freeze-drying and gamma-irradiation are techniques currently used for processing tendon allografts. However, it is still unknown how these processing methods affect graft remodeling. In this study, we used a rat patellar tendon transplantation model to investigate the effect of various processing methods on remodeling by quantifying loss of collagen labeled with a radioactive isotope. The grafts were divided into the following four groups according to the processing method: fresh-frozen, freeze-dried, fresh-frozen and gamma-irradiated, or freeze-dried and gamma-irradiated. The percentage of donor collagen, calculated from hydroxyproline content and radioactivity level, was used as an indicator of graft remodeling. At 2 weeks, the level of donor collagen in the fresh-frozen group was 62%; in the freeze-dried group, 59%; in the fresh-frozen and irradiated group, 57%; and in the freeze-dried and irradiated group, 44%. At 4 weeks, the percentage of donor collagen remaining in grafts decreased to 38% in the fresh-frozen group, 19% in the freeze-dried group, 27% in the fresh-frozen and irradiated group, and 12% in the freeze-dried and irradiated group. Finally, at 12 weeks, the levels were 19% in the fresh-frozen group, 20% in the freeze-dried group, 15% in the fresh-frozen and irradiated group, and 6% in the freeze-dried and irradiated group. The percentages of donor collagen in the freeze-dried and the fresh-frozen and irradiated groups were significantly lower than that in the fresh-frozen group at 4 weeks. The values for the freeze-dried and irradiated group were significantly lower than those for the fresh-frozen and irradiated group at 4 and 12 weeks. These data suggest that freeze-drying, freeze-drying followed by gamma-irradiation, and fresh-freezing followed by gamma-irradiation temporarily accelerate graft remodeling.
In an effort to clarify the responses of a wide range of plant cells to freezing, we examined the responses to freezing of the cells of chilling-sensitive and chilling-resistant tropical and subtropical plants. Among the cells of the plants that we examined, those of African violet ( Saintpaulia grotei Engl.) leaves were most chilling-sensitive, those of hypocotyls in mungbean [ Vigna radiata (L.) R. Wilcz.] seedlings were moderately chilling-sensitive, and those of orchid [ Paphiopedilum insigne (Wallich ex Lindl.) Pfitz.] leaves were chilling-resistant, when all were chilled at -2 degrees C. By contrast, all these plant cells were freezing-sensitive and suffered extensive damage when they were frozen at -2 degrees C. Cryo-scanning electron microscopy (Cryo-SEM) confirmed that, upon chilling at -2 degrees C, both chilling-sensitive and chilling-resistant plant cells were supercooled. Upon freezing at -2 degrees C, by contrast, intracellular freezing occurred in Saintpaulia leaf cells, frost plasmolysis followed by intracellular freezing occurred in mungbean seedling cells, and extracellular freezing (cytorrhysis) occurred in orchid leaf cells. We postulate that chilling-related destabilization of membranes might result in the loss of the ability of the plasma membrane to act as a barrier against the propagation of extracellular ice in chilling-sensitive plant cells. We also examined the role of cell walls in the response to freezing using cells in which the plasma membrane had been disrupted by repeated freezing and thawing. In chilling-sensitive Saintpaulia and mungbean cells, the cells with a disrupted plasma membrane responded to freezing at -2 degrees C by intracellular freezing. By contrast, in chilling-resistant orchid cells, as well as in other cells of chilling-resistant and freezing-resistant plant tissues, including leaves of orchard grass ( Dactylis glomerata L.), leaves of Arabidopsis thaliana (L.) Heynh. and cortical tissues of mulberry ( Morus bombycis Koids.), cells with a disrupted plasma membrane responded to freezing by extracellular freezing. Our results indicate that, in the chilling-sensitive plants cells that we examined, not only the plasma membrane but also the cell wall lacked the ability to serve as a barrier against the propagation of extracellular ice, whereas in the chilling-resistant plant cells that we examined, not only the plasma membrane but also the cell wall acted as a barrier against the propagation of extracellular ice. It appears, therefore, that not only the plasma membrane but also the cell wall greatly influences the freezing behavior of plant cells.
PURPOSE: This study was initiated to determine the negative effect (cryodamage) on human spermatozoa after freeze-thawing and to find out whether freezing of spermatozoa with a computerized biological freezer is more advantageous than freezing above static liquid nitrogen vapour with regard to spermatozoa vitality, chromatin normality, morphology, and membrane integrity. METHODS: Forty-four semen samples were obtained from patients attending andrology laboratory, and each sample was divided into two aliquots. One aliquot was frozen using static liquid nitrogen vapour (G.II) and the second with a computerized biological freezer (G.III). Acridine orange was used for assessment of chromatin cryoinjury, whereas the morphology was evaluated according to WHO criteria. Hypoosmotic swelling test was used to identify membrane integrity and eosin-nigrosin staining was used to determine the vitality of spermatozoa. RESULTS: The mean percentage of normally condensed chromatin in the native semen sample (G.I) decreased significantly (p < .001) after freeze-thawing by using either liquid nitrogen vapour (G.II), or a biological freezer (G.III), which was significantly higher (p < .001) after freezing with liquid nitrogen vapour than after freezing with the biological programmed freezer. Morphologically normal spermatozoa decreased significantly (p < .001) in both freezing methods in comparison to the native semen samples. In addition, membrane integrity of spermatozoa (HOS-test positive) was significantly lower (p < .001) after the freeze-thawing procedure in G.II and G.III compared to G.I. In both these parameters the deterioration was similar among the two freezing procedures. Finally the mean percentage of live spermatozoa decreased significantly (p < .001) in both freezing techniques in relation to the mean value in the neat semen samples. CONCLUSIONS: Freeze-thawing procedure has a detrimental effect on chromatin, morphology, membrane integrity, and vitality of human spermatozoa not only by freezing above static liquid nitrogen vapour but even by using a computerized biological freezer. However, the chromatin deterioration rates are significantly higher by freezing above static liquid nitrogen vapour in comparison to freezing with a programmed biological freezer. Therefore, we recommend the use of this technique for freezing semen especially when ICSI technique is considered as the main therapeutic procedure.
Arabidopsis thaliana (L.) Heynh. has been described as a freezing-tolerant species based on freezing-resistance assays. Nonetheless, this type of experiment does not discriminate between freezing-tolerance and freezing-avoidance mechanisms. The purpose of this paper was to determine which of these two freezing-resistance mechanisms is responsible for freezing resistance in A. thaliana. This was achieved by comparing the thermal properties (ice-nucleation temperature and the freezing temperature) of leaves and the lethal temperature to 10, 50 and 90% of the plants (LT10, LT50, and LT90, respectively). Two wild-type genotypes were used (Columbia and Ler) and their mutants (esk-1 and frs-1, respectively), which differ in their freezing resistance. This study's results indicated that the mutant esk-1, described as a freezing-tolerant species showed freezing tolerance only after a cold-acclimation period. The mutant frs-1, described as freezing sensitive, presented freezing avoidance. Both wild genotypes presented LT50 similar to or higher than the ice-nucleation temperature. Thus, the main freezing-resistance mechanism for A. thaliana is avoidance of freezing by supercooling. No injury of the photosynthetic apparatus was shown by measuring the maximal photochemical efficiency (Fv/Fm) and pigments (chlorophyll and carotenoid) during cold acclimation in all genotypes. During cold acclimation, Columbia and esk-1 increased total soluble carbohydrates in leaves. esk-1 was the only genotype that presented freezing tolerance after cold acclimation. This feature could be related to an increase in sugar accumulation in the apoplast.
The longissimus muscles (LM) from 12 lambs (eight = callipyge [CLPG] and four = normal [NML]; 48 kg) were used to 1) assess the effect of freezing with or without prior immersion in liquid nitrogen on calpastatin activity (CA) and on Warner-Bratzler shear force (WBS) in CLPG, 2) determine the freezing time required to reduce CA and WBS of CLPG to that of NML, and 3) compare sensory panel ratings for CLPG that is aged fresh to CLPG that is aged after freezing. At 24 h postmortem, chops (.64 and 2.54 cm) were removed from each CLPG LM and randomly assigned within side to one of six freezing times (0, 2, 4, 8, 20, and 42 d). Left-side chops were vacuum-packaged and frozen at -20 degrees C for the assigned time (FROZEN) and right-side chops were treated similarly, except that they were frozen in liquid nitrogen first (FLASH). After freezing for the assigned time, the .64-cm chop was immediately assayed for CA, and 2.54-cm chop was thawed, aged at 2 degrees C for 14 d, and refrozen for subsequent WBS measurement. Chops (.64 and 2.54 cm) were also removed from the longissimus of NML lamb carcasses. The d-0 CLPG and NML chops (2.54 cm) were vacuum-packaged, aged at 2 degrees C for 14 d, and frozen for subsequent WBS measurement. Calpastatin activity did not differ between freezing treatments (P = .99) or with the freezing treatment x freezing time interaction (P = .80). Freezing reduced (P = .01) CA in CLPG by 44% from d 0 to d 42. Calpastatin activity for CLPG was similar (P > .05) to that of NML lamb after freezing for 8, 20, or 42 d. Freezing at -20 degrees C for 42 d and then aging for 14 d reduced (P = .01) WBS in CLPG by 44% from d-0 values. Shear force values for CLPG-FROZEN were similar (P > .05) to NML after 8, 20, or 42 d of freezing. Sensory panel tenderness scores were higher (P < .05) for CLPG aged after freezing 42 d than for those aged fresh. Juiciness and flavor ratings did not differ (P > .05) between CLPG aged fresh or after freezing. Freezing for at least 8 d before aging seems to be a viable method for increasing the tenderness of CLPG LM without reducing juiciness or flavor ratings.