Freezing tolerance in relation to cooling rate in an adult insect

In the adult tenebrionid beetle Upis ceramboides unusually low cooling rates are required to demonstrate maximum freezing tolerance, and a very slight change in rate can reduce survival from 100 to 0%. Freezing to ?50 °C results in 100% mortality at rates above 0.35 °C/min, but no injury is apparent if the cooling rate is 0.28 °C/min. The lower lethal temperature, determined with a cooling rate of 0.17 °C/min, is about ?60 °C. The maximum cooling rate which allows full survival is nearly identical to optimal cooling rates previously found for mouse embryos and some lymphocytes, but the striking sensitivity to very slight changes in rate is unique to Upis. Most studies dealing with insect freezing tolerance have utilized rates of 1 °C/min or faster, and the failure of some of these laboratory studies to observe freezing survival may be due to the use of lethal cooling rates.     

Interaction of rate of latent heat removal during freezing and rates of subsequent cooling and warming on survival of the blood protozoan, Babesia rodhaini

Babesia rodhaini parasites in murine blood containing 1.5 m DMSO were frozen at two rates, as judged by the duration of the “freezing plateau”, then cooled to ?196 °C and rewarmed at two rates to detect interactions between the duration of the plateau and rates of subsequent cooling and rewarming. Infectivity tests showed that fast and slow freezing (plateau times of about 1 sec and 30 sec, respectively) had similar effects on parasite survival when cooling was at 130 °C/min and warming was at 800 °C/min. However, when either the cooling rate was increased to 3500 °C/min or the warming rate was decreased to 2.3 °C/min, fast freezing decreased parasite survival more than did slow freezing. It is suggested that fast freezing accentuated the damaging effects of fast cooling and slow warming by increasing intracellular ice formation.     

An experimental study on the freezing of red blood cells with and without hydroxyethyl starch

Red blood cells were frozen in small capillaries down to ?196 °C at different linear cooling rates with or without the cryoadditive HES; the thawing rate was 3000 or 6500 °C/min. Hematocrit and hydroxyethyl starch concentration varied independently. The hemolysis of red blood cells was determined photometrically after 250-fold dilution and compared to totally hemolyzed samples. The typical U-shaped curves for hemolysis as a function of the cooling rate were obtained for all cell suspensions investigated. Relative optimum cooling rates were determined for the respective combinations of HES and hct. The results show that increasing hct causes an increased hemolysis; increased HES concentration C_HES reduces the optimum cooling rate B_opt; increased hct results in higher optimal cooling rates. The findings allow one to establish a linear correlation of the HES concentration and the optimum cooling rates when the dilution of the extracellular medium by the cell water efflux during freezing is taken into account. A comparison with results from larger volumes frozen (25 ml) shows that the established relationship between hematocrit, HES concentration, and optimal cooling rate remains valid.     

Survival of Frozen Mycoplasmas

Cooling to -70 C killed a higher percentage of Acholeplasma laidlawii and Mycoplasma mycoides var. capri cells than cooling to -20 C. However, to preserve cell viability for prolonged periods storage at -70 C was much more preferable. The percentage of cells surviving freezing could be increased by increasing the initial cell concentration or by the addition of dimethyl sulfoxide or glycerol as cryoprotective agents. In the presence of 1.5 M of any one of these agents survival rates of up to 100% could be obtained. The optimal cooling rates for maximal survival of A. laidlawii under the experimental conditions tested were 11 C/min for cooling to -20 C and about 15 C/min for cooling to -70 C. Increasing the warming rate during thawing from 0.6 to 67 C/min increased survival by 3 log. Oleic acid enrichment of A. laidlawii membrane lipids, or reduction in the cholesterol content of M. mycoides var. capri membranes, increased the percentage of organisms surviving freezing. Hence, the composition of membrane lipids appears to have a marked influence on the susceptibility of mycoplasmas to freezing injury.     

Comparisons of glucose,sucrose, and dimethyl sulfoxide as cryoprotective agents for Babesia rodhaini, with estimates of survival rates

Comparisons were made between glucose, sucrose, and dimethyl sulfoxide (DMSO) as cryoprotective agents for the hemoprotozoan parasite, Babesia rodhaini, using infectivity for mice as the criterion of survival. Concentrations of the cryoprotectants tested were from 0.1 to 0.5 M for the sugars, and 1.5 to 2.5 M for DMSO. Glucose and sucrose were comparable as cryoprotectants, although glucose reduced infectivity of the parasites slightly more than did sucrose at above-freezing temperatures. When sucrose and DMSO were compared for cryoprotection during cooling to ?196 °C at nominal rates of 5, 100, and 500 °C/min, parasite survival varied with the type and concentration of cryoprotectant, but was higher in blood containing DMSO at all three cooling rates. The percentages of parasites that survived cooling at 100 °C/min and frozen storage in the presence of DMSO ranged from 20 to 36%.     

Parameters of biological freezing damage in simple solutions: Catalase: II. Demonstration of an optimum-recovery cooling-rate curve in a membraneless system

Seeded solutions of catalase in neutral 10 mM potassium phosphate buffer exhibited characteristic rate dependencies for freeze-thaw damage: Damage increased as the cooling rate was increased, and as the warming rate was decreased. The pattern of warming-rate dependence was independent of the prior cooling rate and also of the addition of KCl or of NaCl to the buffer. In contrast, the cooling-rate curve became almost flat upon addition of 0.1 M KCl, suggesting increased damage from concentrating solute at low cooling rates. In the presence of added NaCl, frank optimum-recovery cooling-rate curves were generated. At low NaCl levels (less than 10 mM) the optimum occurred at 0.5 °C/ min; at 27 and 81 mM NaCl, the optimum shifted to 5 and 20 °C/min, respectively. By comparison with KCl, it appears that the major factor causing damage at low cooling rates in NaCl is acidification. The factor causing damage at high cooling rates remains obscure. The argument that it is due to the trapping of the enzyme molecules at interfaces at high dilution, to be subsequently damaged by shearing stress or dehydration during the recrystallization attending slow warming, is mitigated by the finding that inactivation remains a function of the initial enzyme concentration at all cooling rates. The possibility that a particular conformational state is trapped in an unfavorable temperature zone was also considered: Three simple models were formulated, and the relative order of recovery was deduced for the possible sequences of fast and slow cooling and warming. The permutation observed for catalase was inconsistent with any of these three mechanisms, although they may be pertinent for the red cell and other systems. A final possibility, not yet explored, is that rapid cooling causes damage by producing nonequilibrium freezing, with large deviations of pH and/or solute concentration from those expected at equilibrium.     

Cryoprotection of murine lymphocyte subpopulations using a microprocessor-controlled cooling system

Single-cell suspensions of splenic lymphocytes from 5- to 6-month-old C57BL/6 mice were cryopreserved using cooling rates ranging from ?0.25 to ?10.0 °C/min with the microprocessor-controlled cooling system developed in our laboratory. The cells (30 × 10⁶ cells/ml) were suspended in RPMI 1640 containing 10% FCS and 10% DMSO, and a total volume of 1.75 ml was frozen. Fluorescein-diacetate staining identified viable cells in unfrozen controls and frozen-thawed suspensions. Functional capacity was assessed in vitro by the incorporation of [³H]thymidine by dividing cells activated with graded concentrations of the T-lymphocyte mitogens, PHA-P and Con A, and the B-lymphocyte mitogen, LPS. High percentages of viable cells were recovered after cooling at rates ranging from ?0.5 to ?5.0 °C/min, as compared with those of unfrozen control suspensions. Incorporation of [³H]thymidine by T and B cells reached similar levels after cooling at rates ranging from ?0.25 to ?5.0 °C/min, and the percentage incorporation of [³H]thymidine as compared with that of unfrozen cells was 80–100%, except for T lymphocytes activated with PHA-P after cooling at ?5.0 °C/min. The relative response of cell suspensions to T- and B-cell mitogens, the relative mitogenic index, was unchanged from that of unfrozen controls in suspensions cooled at all rates including two (?0.25 and ?10.0 °C/min), which permitted recovery of only 55% of unfrozen cells. The importance of the constant cooling rates and rapid compensation for heat released at the phase change using the microprocessor-controlled system and of precise determinations of cellular viability and function are discussed and related to the apparent protection conferred on subpopulations of murine lymphocytes using cooling rates ranging from ? 0.25 to ?10.0 °C/min.     

Studies on the mechanism of freezing damage to mouse liver. IV. Effects of ultrarapid freezing on structure and function of isolated mitochondria

Mouse liver mitochondria isolated in 0.25 m sucrose were subjected to progressively increasing cooling rates by quench-thaw from liquid nitrogen, isopentane at ?155 °C, and liquid propane at ?185 °C. Structural damage, assessed by electron microscopy and by quantitation of supernatant protein, increased progressively with the cooling rate. Oxidative phosphorylation (with succinate as substrate) was destroyed at all three cooling rates, while acceptorless respiration (succinoxidase) showed a progressive increase with cooling rate, suggesting uncoupling. The succinate cytochrome c reductase system showed no functional damage. Dimethyl sulfoxide, 10–20% by volume, markedly improved structural preservation of the mitochondria, but did not restore oxidative phosphorylation, and further increased the degree of uncoupling.Upon resuspending the mitochondria in 0.15 m KCl prior to quench-thaw, the succinate cytochrome c reductase system displayed an optimal recovery after isopentane quench-thaw, with a sharp decline at still higher cooling rates, as had been encountered in tissue slice experiments, suggesting a compartmental ice-transition in mitochondria over this range of cooling rates. Structurally, however, the KCl-resuspended mitochondria were equally and maximally disrupted by all three quench-thaw procedures. Sixty percent of the mitochondrial protein was extruded into the supernate, far above the levels released from sucrose-suspended mitochondria by quench-thaw and significantly above the 45% released by sonication. Compared to isotonic KCl, isotonic sucrose was thus providing full cryoprotection for the reductase complex and moderate protection for mitochondrial structure. The discrepancies among the several structural and functional indicators of mitochondrial damage leave little possibility that a single compartmental ice-transition, occurring over this range of cooling rates, could provide a coherent explanation for freezing damage to liver mitochondria.     

Effects of cooling rate on seeds exposed to liquid nitrogen temperatures

The effect of cooling rate on seeds was studied by hydrating pea (Pisum sativum), soybean (Glycine max), and sunflower (Helianthus annuus) seeds to different levels and then cooling them to − 190°C at rates ranging from 1°C/minute to 700°C/minute. When seeds were moist enough to have freezable water (> 0.25 gram H₂O/gram dry weight), rapid cooling rates were optimal for maintaining seed vigor. If the seeds were cooled while at intermediate moisture levels (0.12 to 0.20 gram H₂O per gram dry weight), there appeared to be no effect of cooling rate on seedling vigor. When seeds were very dry (< 0.08 gram H₂O per gram dry weight), cooling rate had no effect on pea, but rapid cooling rates had a marked detrimental effect on soybean and sunflower germination. Glass transitions, detected by differential scanning calorimetry, were observed at all moisture contents in sunflower and soybean cotyledons that were cooled rapidly. In pea, glasses were detectable when cotyledons with high moisture levels were cooled rapidly. The nature of the glasses changed with moisture content. It is suggested that, at high moisture contents, glasses were formed in the aqueous phase, as well as the lipid phase if tissues had high oil contents, and this had beneficial effects on the survival of seeds at low temperatures. At low moisture contents, glasses were observed to form in the lipid phase, and this was associated with detrimental effects on seed viability.     

Evaluation of cryoprotectant and cooling rate for sperm cryopreservation in the euryhaline fish medaka Oryzias latipes

Medaka Oryzias latipes is a well-recognized biomedical fish model because of advantageous features such as small body size, transparency of embryos, and established techniques for gene knockout and modification. The goal of this study was to evaluate two critical factors, cryoprotectant and cooling rate, for sperm cryopreservation in 0.25-ml French straws. The objectives were to: (1) evaluate the acute toxicity of methanol, 2-methoxyethanol (ME), dimethyl sulfoxide (Me₂SO), N,N-dimethylacetamide (DMA), N,N-dimethyl formamide (DMF), and glycerol with concentrations of 5%, 10%, and 15% for 60 min of incubation at 4 °C; (2) evaluate cooling rates from 5 to 25 °C/min for freezing and their interaction with cryoprotectants, and (3) test fertility of thawed sperm cryopreserved with selected cryoprotectants and associated cooling rates. Evaluation of cryoprotectant toxicity showed that methanol and ME (5% and 10%) did not change the sperm motility after 30 min; Me₂SO, DMA, and DMF (10% and 15%) and glycerol (5%, 10% and 15%) significantly decreased the motility of sperm within 1 min after mixing. Based on these results, methanol and ME were selected as cryoprotectants (10%) to evaluate with different cooling rates (from 5 to 25 °C/min) and were compared to Me₂SO and DMF (10%) (based on their use as cryoprotectants in previous publications). Post-thaw motility was affected by cryoprotectant, cooling rate, and their interaction (P ? 0.000). The highest post-thaw motility (50 ± 10%) was observed at a cooling rate of 10 °C/min with methanol as cryoprotectant. Comparable post-thaw motility (37 ± 12%) was obtained at a cooling rate of 15 °C/min with ME as cryoprotectant. With DMF, post-thaw motility at all cooling rates was ?10% which was significantly lower than that of methanol and ME. With Me₂SO, post-thaw motilities were less than 1% at all cooling rates, and significantly lower compared to the other three cryoprotectants (P ? 0.000). When sperm from individual males were cryopreserved with 10% methanol at a cooling rate of 10 °C/min and 10% ME with a rate of 15 °C/min, no difference was found in post-thaw motility. Fertility testing of thawed sperm cryopreserved with 10% methanol at a rate of 10 °C/min showed average hatching of 70 ± 30% which was comparable to that of fresh sperm (86 ± 15%). Overall, this study established a baseline for high-throughput sperm cryopreservation of medaka provides an outline for protocol standardization and use of automated processing equipment in the future.     

Evaluation of a Clostridium perfringens Predictive Model, Developed under Isothermal Conditions in Broth, To Predict Growth in Ground Beef during Cooling

Proper temperature control is essential in minimizing Clostridium perfringens germination, growth, and toxin production. The U.S. Department of Agriculture (USDA) Food Safety and Inspection Service (FSIS) offers two options for the cooling of meat products: follow a standard time-temperature schedule or validate that alternative cooling regimens result in no more than a 1-log₁₀ CFU/g increase of C. perfringens and no growth of Clostridium botulinum. A mathematical model developed by Juneja et al. (Food Microbiol. 16:335-349, 1999) may be helpful in determining if the C. perfringens performance standard has been achieved, but this model has not been extensively validated. The objective of this study was to validate the Juneja 1999 model in ground beef under a variety of changing temperature and temperature abuse situations. The Juneja 1999 model consistently underpredicted growth of C. perfringens during exponential cooling of ground beef. The model also underpredicted growth of C. perfringens in ground beef cooled at two different rates. The results presented here show generally good agreement with published data on the growth of C. perfringens in similar products. The model error may be due to faster-than-expected exponential growth rates in ground beef during cooling or an error in the mathematical formulation of the model.     

Intracellular freezing: effect of extracellular supercooling.

Human erythrocytes were frozen on the stage of a cryomicroscope at accurately controlled constant-cooling rates with varying degrees of extracellular supercooling. The formation of intracellular ice was detected by direct observation of the frozen cells through the microscope. A significant coupling effect was determined between the minimum cooling rate necessary to produce intracellular freezing and the extent of supercooling. Increased degrees of extracellular supercooling reduced the range of cooling rates for which water would freeze within the cell. Specific data points were obtained at ΔT_SC = 0, ?5, and ?12 °C for which the corresponding transition cooling rates were respectively ?845, ?800, and ?11 °C/min.An explanation for the occurrence of this phenomenon is presented based on the physiochemical processes that govern the freezing of a cell suspension.     

The effect of cooling and warming rates on the survival of cryopreserved L-cells

A tissue culture assay has been used to measure the survival of murine lymphoma cells (L-cells) after freezing and thawing in the presence of 2 M glycerol or 1.6 M dimethyl sulfoxide. The effect of variations in cooling rate (0.1 to 10.0 °C/min) and warming rate (0.3 to 200 °C/min) were studied. It was found that survival exhibited a peak at the “conventional” combination of slow cooling and rapid warming (~1 and 200 °C/ min, respectively). It was also shown, however, that a second peak of similar magnitude occurred when the cells were cooled and rewarmed at 0.2-0.3 °C/min. These results are interpreted on the basis of current theories of freezing injury, stressing the importance of damage produced by the recrystallization of intracellular ice and by solute loading. The ultraslow rates of cooling and rewarming which produced the second survival peak are practicable for whole organs, and their potential importance for organ cryopreservation is apparent.     

Relative sensitivity of photosynthesis and respiration to freeze-thaw stress in herbaceous species : importance of realistic freeze-thaw protocols

The relative effect of a freeze-thaw cycle on photosynthesis, respiration, and ion leakage of potato leaf tissue was examined in two potato species, Solanum acaule Bitt. and Solanum commersonii Dun. Photosynthesis was found to be much more sensitive to freezing stress than was respiration, and demonstrated more than a 60% inhibition before any impairment of respiratory function was observed. Photosynthesis showed a slight to moderate inhibition when only 5 to 10% of the total electrolytes had leaked from the tissue (reversible injury). This was in contrast to respiration which showed no impairment until temperatures at which about 50% ion leakage (irreversible injury) had occurred. The influence of freeze-thaw protocol was further examined in S. acaule and S. commersonii, in order to explore discrepancies in the literature as to the relative sensitivities of photosynthesis and respiration. As bath cooling rates increased from 1°C/hour to about 3 or 6°C/hour, there was a dramatic increase in the level of damage to all measured cellular functions. The initiation of ice formation in deeply supercooled tissue caused even greater damage. As the cooling rates used in stress treatments increased, the differential sensitivity between photosynthesis and respiration nearly disappeared. Examination of agriculturally relevant, climatological data from an 11 year period confirmed that air cooling rates in the freezing range do not exceed 2°C/hour. It was demonstrated, in the studies presented here, that simply increasing the actual cooling rate from 1.0 to 2.9°C/hour, in frozen tissue from paired leaflet halves, meant the difference between cell survival and cell death.     

Optimal conditions for the preservation of mouse lymph node cells in liquid nitrogen using cooling rate techniques

The factors that affect the survival of mouse lymphocytes throughout a procedure for storage at ?196 °C have been studied both for the improvement of recovery and the possible extension to the mouse system of cell selection by freezing. After thawing, the survival of cells cooled at different rates in dimethyl sulphoxide (DMSO, 5 or 10%, $\text{v}\text{v}$) was assessed from the [³H]thymidine incorporation in response to phytohaemagglutinin and concanavalin A. Before freezing the protection against freezing damage increased with time (up to 20 min) in DMSO (5%, $\text{v}\text{v}$) at 0 °C. Superimposed upon this effect was toxicity due to the DMSO. During freezing and thawing the cooling rate giving optimal survival was 8 to 15 °C/min for cells in DMSO (5%) and 1 to 3 °C/min for DMSO (10%). Omission of foetal calf serum was detrimental. Rapid thawing (>2.5 °C/min) was superior to slow thawing. After thawing dilution at 25 or 37 °C greatly improved cell survival compared with 0 °C; at 25 °C survival was optimal (75%) at a moderate dilution rate of 2.5 min for a 10-fold dilution in FCS (10%, $\text{v}\text{v}$) followed by gentle centrifugation (50g).Dilution damage during both thawing and post-thaw dilution may be due to osmotic swelling as DMSO and normally excluded solutes leave the cell. The susceptibility of the cell membrane to dilution damage may also be increased during freezing. The need to thaw rapidly and dilute at 25 °C after thawing is probably due to a decrease in dilution stress at higher temperatures. Optimisation of dilution procedures both maximised recovery and also widened the range of cooling rates over which the cells were recovered. These conditions increase the possibility of obtaining good recovery of a mixed cell population using a single cooling procedure. Alternatively, if cell types have different optimal cooling rates, stressful dilution may allow their selection from mixed cell populations.     

Experimental studies on supercooling in two Antarctic micro-arthropods

Samples of mites and Collembola which had been acclimated at 5°C and provided with natural foods were cooled at four constant cooling rates: 1, $\text{1}\text{2}$, $\text{1}\text{4}$, $\text{1}\text{8}\text{deg min}{}^{\mathrm{?1}}$ and ca 20 deg min^?1, and their individual supercooling points measured. Frequency distributions of supercooling points comprised not less than 84 (Alaskozetes antarcticus) and 96 (Cryptopygus antarcticus) individuals in each case. Two modal groups were displayed in these distributions, which were widely separated in temperature and termed low group and high group. In Alaskozetes a trough between ?3 and ?4°C was present in the high-group distribution, which may be due to a lack of a certain class of nucleators. The highest temperatures at which animals froze occurred at the slowest cooling rate ($\text{1}\text{8}\text{deg min}{}^{\mathrm{?1}}$), whereas rapid cooling removed the trough to form a single high-group peak. In Cryptopygus, the high groups were narrow and peaked (<2 deg wide) at all cooling rates, with a downward shift of ca 1 deg between the rates $\text{1}\text{8}$ and $\text{1}\text{2}\text{deg min}{}^{\mathrm{?1}}$. Both species showed a trend towards a lower mean low-group supercooling point at faster rates of cooling, but these were not significant. Regressions of cooling rate on individual low-group supercooling points (≥?20°C) for both species showed a significant negative correlation, which did not differ between species. The distribution of the deviations about each rate-defined mean in the low group for each species was skewed to the right, with 88% occurring between ±2 deg of the means. It is suggested that minor deviations (e.g. halving or doubling of the cooling rate) do not affect the resultant supercooling points at non-constant cooling rates, but a rate of 1 deg min^?1 is to be preferred.     

The effect of cooling and warming rate on cortical cell function of glycerolized rabbit kidneys

Experiments previously reported (I. A. Jacobsen, D. E. Pegg, H. Starklint, J. Chemnitz, C. J. Hunt, P. Barfort, and M. P. Diaper, Cryobiology19, 668, 1982) suggested that rabbit kidneys permeated with 2 M glycerol are least damaged during freezing and thawing if they are cooled very slowly (1 °C/ hr). Using similar techniques of glycerolization, cooling, storage at ?80 °C, rewarming, and deglycerolization, active cell function in cortical tissue slices prepared from such kidneys has now been studied. Oxygen uptake, tissue $\text{K}{}^{\mathrm{+}}\text{Na}{}^{\mathrm{+}}$ ratio after incubation, and slice/medium PAH ratio after incubation were measured. Kidneys cooled at 3.1 °C/min and warmed at 4.2 °C/min gave poor results in the previous studies and the lowest levels of cell function in the present experiments. Kidneys cooled at 1 °C/hr exhibited degrees of slice function that were dependent on warming rate: warming at 1 °C/min was better than warming at either 1 °C/hr or c.20 °C/min. These results refine the previously drawn conclusions, (loc cit) and indicate optimal cooling and warming rates for rabbit kidneys containing 2 M glycerol, in the region of 1 °C/hr cooling and 1 °C/min warming. These rates are much lower than have hitherto been used by others for any system. Some implications of these findings are discussed.     

Evaluation of different cooling rates, equilibration periods and diluents for effects on deep-freezing, enzyme leakage and fertility of taurine bull spermatozoa

We studied the effects of 2 diluents (Tris and milk), 4 cooling rates (10°C/30°C to 5°C for 1 or 2 h), 2 equilibration periods (0 and 2 h) and their interactions on the freezability, glutamic oxaloacetic transaminase (GOT) leakage and fertility of frozen-thawed semen in 18 ejaculates from 3 Friesian bulls. The means of pre- and post-freezing motility, GOT leakage and fertility rates (52.81% based on follow up of 267 inseminated cows) were significantly (P<0.01) influenced by the bulls, cooling rates & equilibration periods, but not by diluents or the interactions studied. The mean prefreeze motility of spermatozoa following 1 h of cooling from 10°C to 5°C was significantly lower (60.38%) and that after 2 h of cooling from 30°C to 5°C was higher (72.38%) than 2 h of cooling from 10°C to 5°C (66.57%) or 1 h of cooling from 30°C to 5°C (67.96%). The mean post-thaw motility observed following 2 h of prefreeze cooling was, however, significantly greater (45%) than after 1 h of cooling (35%) for both the initial temperatures. Leakage of GOT pre- and post-freezing was significantly less following 2h of cooling from 30°C to 5°C (17.26 and 27.36 μmole/L) than after 1 h of cooling from either 10°C (19.71 and 30.13 μmole/L) or 30°C (18.95 and 29.58 μmole/L) and 2 h of cooling from 10°C to 5°C (21.43 and 34.48 μmole/L). The conception rates for semen frozen at the above cooling rates (66.13, 48.65, 56.67 and 42.25%, respectively) were inverse to GOT leakage. An equilibration period of 2 h over that of 0 h at 5°C adversely affected the prefreeze motility and GOT leakage, but it significantly improved postthaw motility (44.03 vs 35.49%) and fertility rates (57.86 vs 47.24%). These findings suggested that both Trisand milk-based diluents were equally efficacious for cryopreservation of bovine semen, and that slow cooling of semen straws over a period of 2 h from 30°C to 5°C as compared with faster cooling rates or a lower initial temperature (10°C), plus at least 2 h of equilibration time at 5°C were essential for optimal freezability, lower enzyme leakage & higher fertility rates within the tropics.     

A comparative study of the morphology and viability of hyphae of Penicillium expansum and Phytophthora nicotianae during freezing and thawing

The changes in morphology of Penicillium expansum Link and Phytophthora nicotianae Van Breda de Haan during freezing and thawing in a growth medium with and without the cryoprotective additive glycerol were examined with a light microscope fitted with a temperature-controlled stage. Viability of 0.5-1.0 mm diameter colonies of both fungi was determined after equivalent rates of cooling to -196 degrees C in the presence or absence of glycerol. In P. expansum shrinkage occurred in all hyphae at rates of cooling of less than 15 degrees C min-1; at faster rates intracellular ice nucleation occurred. The addition of glycerol increased the rate of cooling at which 50% of the hyphae formed intracellular ice from 18 degrees C min-1 to 55 degrees C min-1. This species was particularly resistant to freezing injury and recovery was greater than 60% at all rates of cooling examined. At rapid rates of cooling recovery occurred in hyphae in which intracellular ice had nucleated. In contrast, during the cooling of Ph. nicotianae in the growth medium, shrinkage occurred and no samples survived on thawing from -196 degrees C. However, on the addition of glycerol, shrinkage during freezing decreased and viable hyphae were recovered upon thawing; at rates of cooling over 10 degrees C min-1 the loss of viability was related to glycerol-induced osmotic shrinkage during cooling rather than to the nucleation of intracellular ice.     

Role of cytoplasmic proteins in cold shock injury of Amoeba

Strains of Amoeba have been used to study the mechanisms of cellular injury induced by rapid cooling (cold shock). Cell viability was found to depend on the time and temperature of cold exposure, on the rate of cooling and on the morphology of the cells prior to chilling. All strains underwent a granuloplasmic contraction following undercooling to ?10 °C, although its extent varied; strains most damaged by cold shock exhibited the most violent cytoplasmic contractions. Cryomicroscopy demonstrated that the cellular contraction occurred upon rewarming, not during cooling. Cells damaged by cold shock were osmotically responsive, demonstrating that irreversible damage to the plasmalemma does not account for the phenomenon.Several compounds protected Amoeba against cold shock injury, glycerol and glucose being the most effective. With glycerol an optimum rate of cooling was observed upon cooling to ?10 °C, at both faster and slower cooling rates damage increased.The state of cellular actin in control cells and following cold shock was monitored by the DNase 1 inhibition assay and by electron microscopy. A comparatively “cold shock resistant” strain of A. proteus was found to contain less total actin per unit cellular protein than the more “sensitive” Amoeba sp. strain Bor. In the Bor strain a cold-induced aggregation of cytoplasmic filaments was evident in electron micrographs, presumably a crosslinking of preexisting F-actin.