Protection kinetics of additives on survival of lyophilized bacteria

Samples of washed suspension of Serratia marcescens were frozen at 1- to 4-sec intervals after mixing with solutions of materials considered to act as protective additives. The frozen samples were lyophilized, stored 4 days, and reconstituted with distilled water. Under these conditions, few cells survived without additives, and maximal survival was established 1 sec after mixing, which was the shortest time interval tested. The increased survival of bacteria is assumed to be either a result of some action of the additives at or near the cell surface or of changing the physical properties of the medium, because maximum protection was conferred so rapidly. Significant amounts of sucrose could have entered the cell only if the molecules diffused freely through the cell wall and membrane.The number of organisms surviving lyophilization and exposure to air was found to be dependent upon the interval between mixing and freezing of the sample (3). In conventional systems it is difficult to remove and freeze samples less than 30 sec after mixing two fluids. However, by using a dynamic system, samples can be frozen within 1 sec after cell suspensions are mixed with solutions of various substances. By studying survival of bacteria after they were mixed with protective additives we hoped to determine whether protective effects were caused by a coating of the cells, whether membrane penetration was required, or whether the process might consist of more than one mechanism. This report describes results of experiments in which the contact time before freezing was varied from 1 to 4 sec.     

The effect of thawing velocity on survival and acrosomal integrity of ram spermatozoa frozen at optimal and suboptimal rates in straws

The effect of various thawing velocities on the motility and acrosomal maintenance of ram spermatozoa frozen at 20 degrees C/min (optimal) or 2 degrees C/min (suboptimal) was studied. The freeze-thaw motility and the percentage of intact acrosomes of spermatozoa frozen at 20 degrees C/min increased progressively with the thawing velocity. In semen frozen at 2 degrees C/min, motility of spermatozoa and the percentage of intact acrosomes declined drastically when the thawing velocity obtained in air at 20 degrees C was increased by thawing in water at 20 degrees C. Thawing at higher temperatures markedly increased both motility and acrosomal preservation, but the best results with semen frozen at 2 degrees C/min were lower than those obtained with semen frozen at 20 degrees C/min. The optimal freeze-thaw conditions for semen protected by 4% glycerol were freezing at 20 degrees C/min and thawing in water at 60 or 80 degrees C for 8 or 5 sec, respectively. Semen collected from rams exposed to a decreasing photoperiod exhibited higher motility after freezing and thawing than those exposed to an increasing photoperiod. However, there was no effect on acrosomal preservation after freezing at 20 degrees C/min.     

新鲜土壤样品储存温度和时间对不同形态氮素的影响

土壤氮素形态及含量具有重要的生态学研究意义,而土壤样品的储存对土壤氮素含量的准确测定有很大影响.为了选择合理的土壤样品储存方法,本研究以福建省建瓯市万木林保护区罗浮栲林土壤为研究对象,测定在不同温度(25、4和-20 ℃)、不同储存时间(0、7和30 d)下土壤铵态氮、硝态氮、总氮、可溶性有机氮、氨基酸氮含量和微生物生物量氮,以及冷冻后常温培养过程中的氮素含量.结果表明: 在7 d的储存时间内,除氨基酸氮以外,常温培养样品下其余的氮素含量均有所增加;与新鲜样品相比,冷藏、冷冻样品的所有氮素含量之间均无显著性差异,且氮素含量变化较常温培养下更加稳定.因低温储存样品有刺激氮矿化的效果,在30 d储存时间内,与新鲜样品相比,除可溶性有机氮外,冷藏、冷冻样品的所有氮素含量均显著升高;两种冷储存方法之间无显著差异.因此,新鲜样品带回实验室后应及时处理;如需要冷储藏,时间不要超过半个月.如果需要较长的储存时间,则需将样品放置于更低的温度(-40或-80 ℃).在对储存土壤样品进行培养试验之前,需要进行预培养处理.在预培养过程中,除硝态氮含量呈现先下降再迅速升高的趋势外,其余氮素均随着培养时间逐渐趋近于新鲜土壤样品含量,在培养一周左右恢复到与新鲜土壤样品氮含量最为接近的状态.结合已有研究,对野外取样和风干样品需要5～14 d的预培养,冷储存样品预培养时间不应少于一周.     

Effect of osmolality of skim-milk diluents and thawing rate on cryosurvival of ram spermatozoa

The effect of osmolality of skim-milk diluents (200, 320, 450, 600, and 750 mOsm/kg water) on the survival of ram spermatozoa frozen in straws were investigated after thawing in 39 °C water or in 20 °C air.Spermatozoa motility improved with increasing osmolality of the freezing diluent, irrespective of thawing rate. Diluents of 600 and 750 mOsm resulted in highest motility immediately after thawing and after 60 min incubation at 39 °C. A significant decrease in spermatozoa motility was observed when straws were thawed at 20 °C air with the magnitude of decrease inversely related to osmolality of the freezing diluent. Fertility of progestagen synchronized ewes inseminated with semen frozen in the 600 mOsm hypertonic skim-milk diluent was comparable to that obtained with fresh semen.     

The Effects of Freezing on Faecal Microbiota as Determined Using MiSeq Sequencing and Culture-Based Investigations

Background

High-throughput sequencing has enabled detailed insights into complex microbial environments, including the human gut microbiota. The accuracy of the sequencing data however, is reliant upon appropriate storage of the samples prior to DNA extraction. The aim of this study was to conduct the first MiSeq sequencing investigation into the effects of faecal storage on the microbiota, compared to fresh samples. Culture-based analysis was also completed.

Methods

Seven faecal samples were collected from healthy adults. Samples were separated into fresh (DNA extracted immediately), snap frozen on dry ice and frozen for 7 days at -80°C prior to DNA extraction or samples frozen at -80°C for 7 days before DNA extraction. Sequencing was completed on the Illumina MiSeq platform. Culturing of total aerobes, anaerobes and bifidobacteria was also completed.

Results

No significant differences at phylum or family levels between the treatment groups occurred. At genus level only Faecalibacterium and Leuconostoc were significantly different in the fresh samples compared to the snap frozen group (p = 0.0298; p = 0.0330 respectively). Diversity analysis indicated that samples clustered based on the individual donor, rather than by storage group. No significant differences occurred in the culture-based analysis between the fresh, snap or -80°C frozen samples.

Conclusions

Using the MiSeq platform coupled with culture-based analysis, this study highlighted that limited significant changes in microbiota occur following rapid freezing of faecal samples prior to DNA extraction. Thus, rapid freezing of samples prior to DNA extraction and culturing, preserves the integrity of the microbiota.     

DNA replication in eucaryotic nuclei. Evidence suggesting a specific model of replication

Preimplantation-stage mouse embryos suspended in dimethyl sulfoxide (DMSO) have been used as a model to study details of the response of a simple multicellular system to freezing and thawing. Rapid freezing to ?196 °C kills the embryos unless they have first been cooled very slowly to at least below ?50 °C. The survival of both 2-cell and 8-cell embryos has been found to depend as critically on the rate at which the frozen embryos were thawed as on the rate at which they were first frozen. The damaging consequences of thawing frozen embryos too rapidly have been shown to occur between ?70 and ?20 °C. Finally, the survival of embryos as a function of the time in DMSO prior to freezing and thawing has been compared with their volume changes as a function of time in DMSO. This comparison leads to the tentative conclusion that dimethyl sulfoxide need not permeate the embryos to protect them against freezing damage. Overall, the embryos' response to freezing and thawing is qualitatively similar to that displayed by many other cell types.     

Zona fracture damage and its avoidance during the cryopreservation of mammalian embryos

Although fracture damage to the zonae pellucidae and blastomeres is frequently observed after the cryopreservation of mammalian embryos, little is known of the mechanism by which this occurs. The incidence of damage to zonae was measured when bovine ova with normal zonae were frozen in straws or glass test tubes by standard embryo cryopreservation procedures that yield high rates of survival. Ova were examined for zona damage after warming by procedures that ought to produce little or no thermal stress (slow warming in 20 degrees C air) or high levels of stress (rapid warming in liquid baths). Ova frozen in straws exhibited no zona damage after slow warming at 150 degrees C/min in air (n = 206). However, the incidence of zona damage increased when the straws were warmed rapidly in 20 degrees C (n = 157) or 36 degrees C (n = 159) water (17 and 24%, respectively). Ova in straws warmed rapidly in nonaqueous liquids (ethylene glycol, or silicone oil) exhibited lower rates of zona damage (2 to 5%). Ova frozen in glass tubes exhibited a much higher incidence of zona damage than those frozen in straws, regardless of the warming conditions. Thus, 30% of 114 ova exhibited damage when tubes were warmed slowly at 25 degrees C/min in air, while 54% of 98 ova showed zona damage when tubes were warmed rapidly at 500 degrees C/min in 36 degrees C water. These results are consistent with the view that zona damage is associated with thermally-induced fracturing of the suspension during rapid changes of temperature.     

Duration of thawing on post thaw acrosome morphology and motility of boar spermatozoa frozen in 5-ml maxi-straws

Ejaculates from six boars were frozen in 5-ml maxi-straws and stored in liquid nitrogen. Maxi-straws were thawed by submersion in a 52 degrees C circulating waterbath for 28, 34, 40, 46, 52, or 58 sec. The post thaw percentages of motile sperm and acrosomes with a normal apical ridge (NAR) at the initial microscopic examination were significantly higher (P < 0.05) for maxi-straws submerged for 40 sec than for any other duration of submersion. The mean core temperature of maxi-straws after 40 sec of submersion was 4.8 degrees C. Based on regression equations for post thaw motility and NAR acrosomes, a 37 sec thawing time would be expected to provide the maximal post thaw survival for boar sperm frozen in maxi-straws.     

Sucrose dilution of glycerol from mouse embryos frozen rapidly in liquid nitrogen vapour

The toxic effects of sucrose and the conditions of in-straw glycerol removal after freezing and thawing were studied using Day-3 mouse embryos. At 20 degrees C, exposure to less than or equal to 1.0 M-sucrose for periods up to 30 min had no adverse effects on freshly collected embryos. At 25 and 36 degrees C, however, greater than or equal to 1.0 M-sucrose significantly reduced the developmental potential (P less than 0.001). In the freezing experiments the embryos were placed in 0.5 ml straws containing 40 microliters freezing medium separated by an air bubble from 440 microliters sucrose solution. The straws were frozen rapidly in the vapour about 1 cm above the surface of liquid nitrogen. The post-thaw viability was substantially better after sucrose dilution at 20 degrees C than at 36 degrees C. Mixing the freezing medium with the sucrose diluent immediately after thawing further improved the rate of survival relative to mixing just before freezing (P less than 0.001). The best survival was obtained when the freezing medium contained 3.0 M-glycerol + 0.25 M-sucrose; it was mixed with the diluent after thawing and the glycerol was removed at 20 degrees C. Under such conditions the sucrose concentration in the diluent had no significant effect on the rate of development (0.5 M, 69%; 1.0 M, 73%; 1.5 M, 64%). The results show that during sucrose dilution the temperature should be strictly controlled and suggest that intracellular and extracellular concentrations of glycerol are important in the cryoprotection of embryos.     

Improving the Prediction of Prostate Cancer Overall Survival by Supplementing Readily Available Clinical Data with Gene Expression Levels of IGFBP3 and F3 in Formalin-Fixed Paraffin Embedded Core Needle Biopsy Material

Background

A previously reported expression signature of three genes (IGFBP3, F3 and VGLL3) was shown to have potential prognostic value in estimating overall and cancer-specific survivals at diagnosis of prostate cancer in a pilot cohort study using freshly frozen Fine Needle Aspiration (FNA) samples.

Methods

We carried out a new cohort study with 241 prostate cancer patients diagnosed from 2004–2007 with a follow-up exceeding 6 years in order to verify the prognostic value of gene expression signature in formalin fixed paraffin embedded (FFPE) prostate core needle biopsy tissue samples. The cohort consisted of four patient groups with different survival times and death causes. A four multiplex one-step RT-qPCR test kit, designed and optimized for measuring the expression signature in FFPE core needle biopsy samples, was used. In archive FFPE biopsy samples the expression differences of two genes (IGFBP3 and F3) were measured. The survival time predictions using the current clinical parameters only, such as age at diagnosis, Gleason score, PSA value and tumor stage, and clinical parameters supplemented with the expression levels of IGFBP3 and F3, were compared.

Results

When combined with currently used clinical parameters, the gene expression levels of IGFBP3 and F3 are improving the prediction of survival time as compared to using clinical parameters alone.

Conclusion

The assessment of IGFBP3 and F3 gene expression levels in FFPE prostate cancer tissue would provide an improved survival prediction for prostate cancer patients at the time of diagnosis.     

The freezing threshold of peripheral motor nerves following pretreatment with dimethyl sulfoxide and ethanol: An electrophysiologic and light and electron microscopic study on the sciatic nerve of rabbits

The right sciatic nerve of 78 cross-breed rabbits was exposed. Fifty-two of these nerves were treated with 10% DMSO or 10% ethanol for a period of 10 min before being frozen or supercooled. Twenty-six nerves were just supercooled or frozen, these being used for control purposes. A capacity-limited solid silver probe, 15 cm in length and 1 cm in diameter, was employed. Ethanol was used as the cooling agent. The freezing or supercooling temperatures were 0, ?5, ?10, ?20, ?25, and ?30 °C, and the freezing or supercooling times were 10, 30, and 120 sec. One, two, or four freeze- or supercool-thaw cycles were employed. After electric supramaximal stimulation with 3.8 V, the amplitudes of the action potentials (AP) were measured before and immediately after 1, 3, 5, 10, 20, 30, 60, and 90 min and 2, 5, and 10 days after supercooling or freezing, respectively. The pretreated nerves were examined under light and electron microscopes after 2 days. The damage to the nerve fibers depends on the freezing or supercooling temperature, the freezing or supercooling time, and the number of freeze- or supercooling-thaw cycles. Electrophysiologically, this damage leads to a decrease in the amplitude or complete disappearance of the APs and to a reduction in motor function. The morphological findings were clumping and at times even vacuolization of the myelin sheaths and a thickening of the axon with a loss of microfilaments, microtubules, and mitochondria. First the large, then the medium and small myelinated nerve fibers appeared to be affected. The unmyelinated fibers seem well preserved. No differences in quality but differences in quantity were observed between those nerves treated with cryoprotective agents and the nontreated control nerves. With the latter, the damage was spread diffusely over the whole nerve; whereas with the pretreated nerves, damage was localized in the periphery, primarily where the cryoprobe was applied.DMSO and ethanol have a cryoprotectivc effect on the nerves, and in this respect it would appear, from electron microscopic observations, that fewer nerve fibers were damaged compared with the control nerves and, from an electrophysiological viewpoint, following pretreatment the action potentials had a greater amplitude than that of the control nerves.After pretreatmcnt with 10% DMSO or 10% ethanol, the freezing or supercooling threshold of the sciatic nerves was determined in relation to the freezing or supercooling times and the freeze- or supercool-thaw cycles. With one freeze-thaw cycle this freezing threshold was, for both 10% DMSO and 10% ethanol, ?25 °C with a freezing time of 10 sec, ?20 °C with a freezing time of up to 30 sec, and ?15 °C with a freezing time of up to 120 sec. Consequently, the freezing threshold was higher than with motor nerves frozen under the same conditions without cryoprotective agents (the controls).If these experimental results could be applied in clinical cryosurgery it might be possible to preserve a peripheral motor nerve in the periphery of the cryolesion to a certain extent by injecting such cryoprotective agents around the nerve.     

Preservation of Serratia marcescens by High-Vacuum Lyophilization

Water-washed Serratia marcescens (ATCC strain 14041) cells were lyophilized in an all-glass system capable of evacuation to pressures of less than 5 x 10(-6) torr. Lyophilization at the lowest pressures resulted in 50 to 65% survival for unstabilized washed organisms compared with 10 to 20% when the cells were lyophilized at pressures of about 2.5 x 10(-2) torr. At the latter pressures, 45 to 65% survival was obtained when NaCl or Naylor-Smith stabilizer was added to the cell suspensions before lyophilization. However, the stabilizers failed to increase significantly the levels of survival compared with water suspension when cells were lyophilized at pressures less than 10(-5) torr. The high survival rates obtained by the high-vacuum technique may be attributed to the reduction of traces of molecular oxygen which has been reported to be destructive of the dried bacteria. Survival of unstabilized dried S. marcescens after 1-day storage increased markedly with decreasing sealing pressure. Under the highest vacuum attained, survival of the dried bacteria was not impaired by storage for up to 1 month at Dry Ice temperatures; at higher temperatures, viability losses occurred. Exposure of the dried unstabilized bacteria to dry air resulted in rapid viability loss. The inactivation could be stopped almost immediately by evacuation to pressures of less than 10(-5) torr, but the evacuation failed to reverse the viability losses that occurred during exposure.     

Effect of thawing regimens on the morphofunctional state of canine spermatozoa

The effect of 2 thawing regimens (37 degrees C for 8 sec and 55 degrees C for 5 sec) was followed up on semen parameters related to the viability of canine spermatozoa. The ejaculates were frozen in the form of pellets on dry ice in the following cryoprotective extenders: TRIS-fructose (TF), TRIS-glucose (TG), and sucrose-lactose (SL). For the 3 extenders, significant differences were found in the percentage of motile spermatozoa and their survival rate up to 300 min in favor of the 55 degrees C vs the 37 degrees C thawing regimens. Structural changes such as swelling, breakage and absence of acrosomes were observed in the samples frozen in the 3 cryoprotective extenders. A considerably lower percentage of spermatozoa with damaged acrosomes was recorded at 55 degrees C in comparison with that found at 37 degrees C (P < 0.05 for TG, TF and SL). Enzymocytochemical analysis was made of NADH-tetrazolium reductase activity in thawed spermatozoa. Cells showing moderate and strong intensity of the cytochemical reaction were found after both regimens of thawing. The percentage of spermatozoa manifesting strong intensity of the reaction was comparatively higher after thawing at 55 degrees C (31.8 +/- 2.06) than at 37 degrees C (23.7 +/- 1.41; P < 0.01). The thawing regimens were the factors that exerted influence on the morphofunctional state of frozen canine spermatozoa, irrespective of the cryoprotective extenders used, in the present study. Thus the optimal preservation of sperm viability was achieved by thawing at 55 degrees C for 5 sec.     

Early pituitary LH response to injections of GnRH in ewes

In the deep anoestrous period (June), five intact ewes and five ovariectomized ewes received 50 ug synthetic gonadotrophin-releasing hormone (GnRH). In the mid-breeding season (October), the GnRH administrations were repeated in five intact and four ovariectomized ewes; the former were in the luteal phase of the cycle. Blood samples were collected every 30 sec for 15 min, then at 15-min intervals. Release of luteinizing hormone (LH) occurred as soon as the second minute after injection in all ewes. This early response was earlier and more abrupt in the ovariectomized ewes than in the intact animals. In a second experiment three intact ewes that were in deep anoestrus received 50 ug GnRH followed 5 h 20 min later by a second identical injection. Another three intact ewes in deep anoestrus received two injections of 1 ug GnRH. Blood samples were taken every 15 sec for 15 min, then every 20 min until the next injection, and for a further 5 h after the second injection. This regimen was repeated in mid-breeding season during the luteal phase. There was again a very early release of LH; the magnitude of response was similar after the first injection of either 50 ug or 1 ug GnRH to intact ewes either in the breeding season or during deep anoestrus. However, a greater early release of LH was obtained at the lower dose only after the second injection of GnRH. Apart from this exception, the similar early release of LH occurred in spite of different amounts of LH released thereafter in response to the two doses of GnRH. It is suggested that the early response to GnRH consists of LH stored in a "readily releasable" pool in the pituitary, whereas the main release of LH may be a result of increased synthesis and/or release of a more stable pool.     

Effect of cryoprotectant concentration, equilibration time and thawing procedure on survival and development of rapid frozen-thawed mature mouse oocytes

Experiments were conducted to develop a simple rapid-freezing protocol for mature mouse oocytes that would yield a high proportion of oocytes with developmental potential. The effects of concentration (3.5, 4.5 and 6.0 M dimethyl sulfoxide (DMSO) all with 0.5 M sucrose) and the duration of exposure (2.5 min vs 45 sec) of oocytes to the cryoprotectant and its extraction after thawing in 2, 3 or 4 steps of descending sucrose concentration were studied. The most effective of the rapid-freezing and thawing protocols (4.5 M DMSO; 45 sec exposure and 3-step thawing) was compared to slow freezing protocols using 1.5 M DMSO and 1.0 M 1,2 propanediol as cryoprotectants. The DMSO concentrations had an effect on survival, fertilization and embryo development using short (45 sec) but not long (2.5 min) exposure. The rate of morphological oocyte survival was significantly higher using 4.5 M DMSO than 3.5 or 6.0 M (92% vs 82 and 73%, respectively). The development of fertilized embryos to blastocysts was also significantly higher at 4.5 M than at 3.5 or 6.0 M (68% vs 42 and 53%, respectively). The extraction of cryoprotectant in 3 or 4 steps of descending sucrose concentration resulted in higher survival (P < 0.01) and fertilization than in 2 steps. The best survival, fertilization and development was achieved with the 3-step procedure. Optimal combinations of conditions were 4.5 M DMSO at 45 sec prefreeze exposure and 3-step extraction of the cryoprotectant. Oocytes frozen by conventional methods had a survival, fertilization and development to blastocyst rate significantly lower than those frozen under the optimal rapid conditions. Thus rapid freezing of mature mouse oocytes with 4.5 M DMSO + 0.5 M sucrose and short prefreeze exposure is effective and has the additional advantage of being less time-consuming than slow freezing methods.     

Non-invasive measurement of faecal glucocorticoid metabolites in Upland Geese <Emphasis Type="Italic">Chloephaga</Emphasis><Emphasis Type="Italic">picta</Emphasis>

Glucocorticoid (GC) hormones rise in response to stressors, including natural events including weather or predator presence, and human activities, such as hunting, scientific research or recreational visits. However, because blood sampling itself causes stress and is dangerous or even impossible in some wildlife species, feedback-free methods for GC determination are needed to assess stress in these animals. Faecal GC analyses have thus gained interest. Here, we validate a non-invasive method to estimate the physiological stress in the Upland goose Chloephaga picta. An adrenocorticotropin hormone (ACTH) challenge was conducted in captive adults (female and male), and droppings were collected before, during and after the experiment. Corticosterone metabolite (CM) secretion in response to the ACTH challenge was measured with several enzyme immunoassays (EIA) to find the most appropriate test. We used CM levels during the periods before and after the experiment as control data. An EIA for 11-oxoetiocholanolone achieved the highest response to the ACTH challenge and also reflected a stress response to unfamiliar environment. Furthermore, CM concentrations of dry samples were highly correlated with the corresponding non-dried (frozen) samples. The data suggest that this method is appropriate to measure the stress in Upland geese, and that samples can be stored either frozen or dry form.     

Experimental study of enriched frozen diet on digestive enzymes and growth of juvenile cuttlefish Sepia officinalis L. (Mollusca Cephalopoda)

Hatchlings cuttlefish were reared in the laboratory from hatching until 30 days old, fed with live shrimp, frozen shrimp or fish oil-enriched frozen shrimp. Survival of cuttlefish fed with oil-enriched frozen shrimp was better than in animals receiving live shrimp. However, there was no difference with cuttlefish fed with frozen shrimp, even if survival of those receiving oil-enriched frozen shrimp was always higher all along the experiment. Lower survival in animals fed with live shrimp represented the problem of using such food and confirms the necessity to elaborate an artificial food. Utilization of artemia was detrimental to growth and induced low values of instantaneous growth rate (IGR) and conversion rate even after feeding cuttlefish with shrimp. Nevertheless, growth parameters evolutions generally corresponded to those observed by other researchers. The profile noticed at the end of the experiment is typically observed when cuttlefish acquire their adult digestive system. Main differences were observed between groups fed with live shrimp or oil-enriched frozen shrimp. Enrichment did not induce same growth as in cuttlefish receiving live prey. However, at 20 and 25 days after hatching (DAH), in cuttlefish fed with oil-enriched frozen shrimp, ration was lower for the same growth than in other groups.These data showed capacity of juvenile cuttlefish to adjust their digestive enzyme activities according to the diet and the stage of development. Indeed, chymotrypsin was strongly influenced by enrichment, while other enzymes showed difference between live and frozen preys. Trypsin exhibited regulation by diet after 20 DAH. Freezing seemed to delay development as acid phosphatases, characteristic of first stages of cuttlefish, had lower activity in cuttlefish fed with live shrimp at 10 DAH. Moreover, influence of the stage of development was strong as activities between 20 and 30 DAH were different in all groups. This was in relation with evolution of the digestive system. These data illustrated the difficulty to elaborate optimal diet as digestive system evolves.     

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.     

In vitro assessment of a direct transfer vitrification procedure for bovine embryos

We developed a simple vitrification technique for bovine embryos that could permit direct transfer. Embryos were produced in-vitro by standard procedures. The base medium for cryopreservation was a chemically defined medium similar to SOF + 25 mM Hepes and 0.25% fatty acid free bovine serum albumin (FAF-BSA) (HCDM2). In experiment 1, embryos were first exposed to 3.5M ethylene glycol (V1) for 1, 2 or 3 min at room temperature (20-24 degrees C), and then moved to 7 M ethylene glycol (V2) at 4 or 20-24 degrees C and loaded in 0.25-mL straws. After 45 s in 7 M ethylene glycol, straws were placed in liquid nitrogen. Embryos that were loaded at 20-24 degrees C had higher survival rates than those loaded at 4 degrees C (P<0.05). Exposure for 1 min was best for morulae, while 3 min was best for blastocysts. In experiment 2, blastocysts were handled at 24 degrees C and exposed to two concentrations of ethylene glycol in V1 (3.5 or 5 M) followed by V2 as in experiment 1, two warming temperatures (20 or 37 degrees C) and two post-warming holding times until culture (5 or 15 min). Exposure to 5 M ethylene glycol and warming at 37 degrees C was the optimal combination of procedures, and embryos survived well after 15 min in straws if warmed at 37 degrees C. In experiment 3, ethylene glycol concentration (3, 4 or 5 M) and exposure time (0.5 or 1 min) during two-step addition of cryoprotectant were studied for bovine morulae. In experiment 4, morulae were exposed to V2 for 30 or 45 s in HCDM2 or Vigro holding medium and then held in 22-24 degrees C air or 37 degrees C water post-warming. Experiment 5 was like experiment 4 except blastocysts were used. Overall survival rates of blastocysts in experiment 5 averaged 80% of non-vitrified controls after 48 h culture. The survival rates with in vitro-produced morulae in experiments 1, 3 and 4 were unacceptable. Vitrification solutions based on Vigro tended to result in higher survival than HCDM2 for blastocysts, but not morulae. In experiment 6, the survival rate in vitro of in vivo-produced morulae and blastocysts after two-step vitrification was nearly 100%. Our vitrification technique was very effective for in vitro produced blastocysts, but not for in vitro-produced morulae.     

Rapid cold-hardening in larvae of the Antarctic midge Belgica antarctica: cellular cold-sensing and a role for calcium

In many insects, the rapid cold-hardening (RCH) response significantly enhances cold tolerance in minutes to hours. Larvae of the Antarctic midge, Belgica antarctica, exhibit a novel form of RCH, by which they increase their freezing tolerance. In this study, we examined whether cold-sensing and RCH in B. antarctica occur in vitro and whether calcium is required to generate RCH. As demonstrated previously, 1 h at -5 degrees C significantly increased organismal freezing tolerance at both -15 degrees C and -20 degrees C. Likewise, RCH enhanced cell survival of fat body, Malpighian tubules, and midgut tissue of larvae frozen at -20 degrees C. Furthermore, isolated tissues retained the capacity for RCH in vitro, as demonstrated with both a dye exclusion assay and a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)-based viability assay, thus indicating that cold-sensing and RCH in B. antarctica occur at the cellular level. Interestingly, there was no difference in survival between tissues that were supercooled at -5 degrees C and those frozen at -5 degrees C, suggesting that temperature mediates the RCH response independent of the freezing of body fluids. Finally, we demonstrated that calcium is required for RCH to occur. Removing calcium from the incubating solution slightly decreased cell survival after RCH treatments, while blocking calcium with the intracellular chelator BAPTA-AM significantly reduced survival in the RCH treatments. The calmodulin inhibitor N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7) also significantly reduced cell survival in the RCH treatments, thus supporting a role for calcium in RCH. This is the first report implicating calcium as an important second messenger in the RCH response.