Effect of supercooling and freezing on photosynthesis in freezing tolerant leaves of Afroalpine ‘giant rosette’ plants

Summary The effect of supercooling and freezing on the photosynthetic capability of representatives of the permanent frost hardy giant rosette plants Dendrosenecio keniodendron, D. brassica and Lobelia telekii, of the tropical alpine regions was investigated with the non-invasive chlorophyll a fluorescence technique. While supercooling, normal chlorophyll a fluorescence kinetics exhibiting the sequence 0, I, (D), P, S, M, were recorded, however with some retardation of both, the fast and the slow characteristics as compared to those obtained at day-time temperature. As long as the leaves remained unfrozen, the rise of the variable fluorescence F from the level 0 to P was inversely related to a drop of the temperature from about 0°C to-8°C. The increase of F with lower temperature is understood to result from a decrease of the velocity of the quenching reactions while photoreduction of the primary electron acceptor appeared to be unimpeded. The second fluorescence maximum (M), usually interpreted to indicate the commencement of the biochemical reactions of photosynthesis was consistenly to be observed during supercooling. Fluoescence induction kinetics of frozen leaves showed only fast rise to presumably F _max which was not followed by a significant decay for as long as 4 min. The lack of substantial quenching indicates that in the freeze-dehydrated state neither reoxidation of the primary acceptor nor energetization of the thylakoid membrane was accomplished. This effect however was immediately and fully reserved upon thawing of the leaves when the usual fluorescence induction kinetics as well as normal rates of CO₂-uptake were observed. Thus the permanent frost-hardy afroalpine plants do not exhibit any even short-term memory effect of the nocturnal frost on such a delicate process as is photosynthesis.     

Protective effect of intracellular ice during freezing?

Injury results during freezing when cells are exposed to increasing concentrations of solutes or by the formation of intracellular ice. Methods to protect cells from the damaging effects of freezing have focused on the addition of cryoprotective chemicals and the determination of optimal cooling rates. Based on other studies of innocuous intracellular ice formation, this study investigates the potential for this ice to protect cells from injury during subsequent slow cooling. V-79W Chinese hamster fibroblasts and Madin-Darby Canine Kidney (MDCK) cells were cultured as single attached cells or confluent monolayers. The incidence of intracellular ice formation (IIF) in the cultures at the start of cooling was pre-determined using one of two different extracellular ice nucleation temperatures (-5 or -10 degrees C). Samples were then cooled at 1 degrees C/min to the experimental temperature (-5 to -40 degrees C) where samples were warmed rapidly and cell survival assessed using membrane integrity and metabolic activity. For single attached cells, the lower ice nucleation temperature, corresponding to increased incidence of IIF, resulted in decreased post-thaw cell recovery. In contrast, confluent monolayers in which IIF has been shown to be innocuous, show higher survival after cooling to temperatures as low as -40 degrees C, supporting the concept that intracellular ice confers cryoprotection by preventing cell dehydration during subsequent slow cooling.     

Effects of different extenders on DNA integrity of boar spermatozoa following freezing–thawing

The sperm-rich fraction, collected from eight mature Yorkshire boars, was frozen in an extender containing 9% LDL (w/v), 100 mM trehalose, or 20% yolk (v/v), respectively. Sperm DNA integrity was assessed using the single-cell gel electrophoresis (SCGE). Other sperm quality characteristics such as motility, acrosome and membrane integrity were also monitored. The results showed that freezing–thawing caused an increase in sperm DNA fragmentation, and extender containing 9% LDL could significantly protect sperm DNA integrity (P < 0.05) from the damage caused by cryopreservation and decrease DNA damages compared with extender containing 100 mM trehalose and 20% yolk (v/v). No significant difference in damaged DNA was detected between frozen and unfrozen semen samples for extender of 9% LDL and 100 mM trehalose, but cryopreservation could increase the degree of DNA damage (P < 0.05), the percentage of damaged DNA degree of grade 2 and 3 was significantly increased. The deterioration in post-thaw sperm DNA integrity was concurrent with reduced sperm characteristics. The data here demonstrated that the cryoprotectant played a fundamental role in reducing boar sperm DNA damage and protecting DNA integrity. It can be suggested that evaluation of sperm DNA integrity, coupled with correlative and basic characteristics such as motility, acrosome integrity and membrane integrity, may aid in determining the quality of frozen boar semen.     

Changes in hydraulic conductivity upon freezing of the xylem of Populus x canadensis Moench “robusta”

Summary The impact of freezing stress on the hydraulic conductivity was studied in 4- to 6-year-old branches of Populus x canadensis Moench robusta under gravity flow conditions. In fresh branch segments, the hydraulic conductivity was approximately 3–6×10^-2 1 h^-1kPa^-1m and the specific conductivity approximately 22 1 h^-1kPa^-1m^-1. Depending on the gas content of the solutions fed to the xylem of the segments, their hydraulic conductivity was lowered by a freezing-thawing cycle by 20–50%. However, full recovery of hydraulic conductivity was found after about 2 days. Degassed solutions in contrast showed no impediment to flow after the same treatment. The results give evidence, firstly, that the harmful effect of freezing on functioning of water conducting elements is due to the formation of bubbles in xylem sap containing gas in solution, and secondly, that recovery from this impediment is possible in microporous vessels within a period of about 2 days.     

Effect of Gynostemma Pentaphyllum Polysaccharide on boar spermatozoa quality following freezing–thawing

Gynostemma Pentaphyllum Polysaccharide (GPP) was added at concentrations of 0.25, 0.5, 1.0, 1.5 and 2.0 mg/ml to the extenders used to freeze boar semen and its effects on the quality of frozen–thawed sperm were assessed. The sperm motility was significantly higher in the extenders containing 0.25 and 0.5 mg/ml GPP, as compared to other groups (P < 0.05). The extender supplemented with 0.5 mg/ml GPP favored the highest intact membrane and intact acrosome percentages in comparison with other groups (P < 0.05), respectively. The mitochondrial activity was significantly higher at the concentrations of 0.25, 0.5 and 1.0 mg/ml GPP than that of other treatments, and the control group (P < 0.05). In biochemical assays, the extender supplemented with 0.25 and 0.5 mg/ml GPP significantly improved SOD levels, compared to other groups (P > 0.05). However, the extenders supplemented with GPP did not cause significant differences in levels of CAT and GSH-Px, compared to the control (P > 0.05). In summary, GPP exhibited a dose-related response and the lower concentration produced greater protective effect. According to the standard semen quality parameters and antioxidant activities measured in this study, the concentration of 0.5 mg/ml GPP caused a beneficial cryoprotective effects on the quality of frozen–thawed boar semen. It is proposed that an extender containing 0.5 mg/ml GPP could be used as cryoprotective medium of better efficiency.     

The occurrence of “mixed” nuclear bag intrafusal fibers in the cat muscle spindle

Summary A total of 147 muscle spindles was studied histochemically in serial transverse sections of 42 cat tenuissimus muscle specimens. Nuclear bag₁, nuclear bag₂ and nuclear chain intrafusal muscle fibers were distinguished by the differential staining resulting from the reactions for myosin adenosine 5-triphosphatase and nicotinamide adenine dinucleotide tetrazolium reductase. The majority of intrafusal fibers were of the same histochemical type at both fiber poles. However, seven muscle spindles contained one nuclear bag fiber each that presented as a bag₁ in one pole and as a bag₂ in the other pole. These mixed nuclear bag fibers were found in spindles that also contained at least one bag₁ and one bag₂ fiber of equivalent histochemical presentation in both fiber poles. The mixed bag fibers displayed differences of apparent fiber diameter and relative polar length between the two fiber poles. The motor innervation pattern, as revealed by staining for cholinesterase, was also dissimilar between the two poles of mixed bag fibers. The study indicates that the spindle equatorial region may in some instances serve as a boundary between two morphologically and histochemically different poles of the same intrafusal fiber.     

Comparative freezing patterns of bark and xylem of “Siberian C” and “Redhaven” peach twigs

An electrophoretic mobility technique was used to study freezing patterns in excised peach twigs. Moisture content was the only qualitative difference in initial freezing patterns of similar tissues of Redhaven and Siberian C. Siberian C contained up to 18% less moisture than Redhaven. Major differences in the shape of the transition pattern were detected between bark and xylem. Even though bark tissues had twice the water content of xylem, the bark exhibited equilibrium freezing while the xylem underwent nonequilibrium freezing. Bark water must be intimately associated with the living protoplast, while xylem water is less closely associated with cellular components. Comparison of bark and xylem freezing curves with sucrose and cellulose model systems suggested that bark freezing was similar to the sucrose model while xylem freezing was similar to the cellulose model.     

Effect of freezing–thawing process and quercetin on human sperm survival and DNA integrity

We aimed in the first part of our work to study the effect of cryopreservation on the human sperm DNA integrity and the activation of caspase 3, the main apoptosis indicator. In the second part, we were interested in testing the effect of quercetin, as an antioxidant, in preventing sperm damage during the freeze–thawing process. Seventeen semen samples were obtained from 17 men recruited for infertility investigations. Liquefied sperm was cryopreserved using spermfreeze®. Nine of the used samples were divided into two aliquots; the first one was cryopreserved with spermfreeze only (control) and the second one was cryopreserved with spermfreeze supplemented with quercetin to a final concentration of 50 μM. Sperm motility and viability were assessed according to WHO criteria. We used TUNEL assay and the Oxy DNA assay to assess sperm DNA integrity. Activated caspase 3 levels were measured in spermatozoa using fluorescein-labeled inhibitor of caspase (FLICA). Cryopreservation led to a significant increase in sperm DNA fragmentation, DNA oxidation and caspase 3 activation (p < 0.01). Supplementation of the cryopreservation medium with quercetrin induced a significant improvement in post thaw sperm parameters, compared to those of control, regarding sperm motility (p = 0.007), viability (p = 0.008) and DNA integrity (p = 0.02); however, it had no effect on caspase 3 activation (p = 0.3). We conclude that oxidative stress plays a major role in inducing sperm cryodamage but implication of apoptosis in this impairment requires further investigations. Quercetin could have protective effect during cryopreservation but further research is needed to confirm this effect.     

Genetic variability and QTL mapping of freezing tolerance and related traits in Medicago truncatula

Freezing is a major environmental limitation to crop productivity for a number of species including legumes. We investigated the genetic determinism of freezing tolerance in the model legume Medicago truncatula Gaertn (M. truncatula). After having observed a large variation for freezing tolerance among 15 M. truncatula accessions, the progeny of a F6 recombinant inbred line population, derived from a cross between two accessions, was acclimated to low above-freezing temperatures and assessed for: (a) number of leaves (NOL), leaf area (LA), chlorophyll content index (CCI), shoot and root dry weights (SDW and RDW) at the end of the acclimation period and (b) visual freezing damage (FD) during the freezing treatment and 2 weeks after regrowth and foliar electrolyte leakage (EL) 2 weeks after regrowth. Consistent QTL positions with additive effects for FD were found on LG1, LG4 and LG6, the latter being the most explanatory (R ² ≈ 40 %). QTL for NOL, QTL for EL, NOL and RDW, and QTL for EL and CCI colocalized with FD QTL on LG1, LG4 and LG6, respectively. Favorable alleles for these additive effects were brought by the same parent suggesting that this accession contributes to superior freezing tolerance by affecting plants’ capacity to maintain growth at low above-freezing temperatures. No epistatic effects were found between FD QTL, but for each of the studied traits, 3–6 epistatic effects were detected between loci not detected directly as QTL. These results open the way to the assessment of syntenic relationships between QTL for frost tolerance in M. truncatula and cultivated legume species.     

Effect of ‘in air’ freezing on post-thaw recovery of Callithrix jacchus mesenchymal stromal cells and properties of 3D collagen-hydroxyapatite scaffolds

Through enabling an efficient supply of cells and tissues in the health sector on demand, cryopreservation is increasingly becoming one of the mainstream technologies in rapid translation and commercialization of regenerative medicine research. Cryopreservation of tissue-engineered constructs (TECs) is an emerging trend that requires the development of practically competitive biobanking technologies. In our previous studies, we demonstrated that conventional slow-freezing using dimethyl sulfoxide (Me₂SO) does not provide sufficient protection of mesenchymal stromal cells (MSCs) frozen in 3D collagen-hydroxyapatite scaffolds. After simple modifications to a cryopreservation protocol, we report on significantly improved cryopreservation of TECs.Porous 3D scaffolds were fabricated using freeze-drying of a mineralized collagen suspension and following chemical crosslinking. Amnion-derived MSCs from common marmoset monkey Callithrix jacchus were seeded onto scaffolds in static conditions. Cell-seeded scaffolds were subjected to 24 h pre-treatment with 100 mM sucrose and slow freezing in 10% Me₂SO/20% FBS alone or supplemented with 300 mM sucrose. Scaffolds were frozen ‘in air’ and thawed using a two-step procedure. Diverse analytical methods were used for the interpretation of cryopreservation outcome for both cell-seeded and cell-free scaffolds. In both groups, cells exhibited their typical shape and well-preserved cell-cell and cell-matrix contacts after thawing. Moreover, viability test 24 h post-thaw demonstrated that application of sucrose in the cryoprotective solution preserves a significantly greater portion of sucrose-pretreated cells (more than 80%) in comparison to Me₂SO alone (60%).No differences in overall protein structure and porosity of frozen scaffolds were revealed whereas their compressive stress was lower than in the control group. In conclusion, this approach holds promise for the cryopreservation of ‘ready-to-use’ TECs.     

Equilibrium freezing of leaf water and extracellular ice formation in Afroalpine ‘giant rosette’ plants

The water potentials of frozen leaves of Afroalpine plants were measured psychrometrically in the field. Comparison of these potentials with the osmotic potentials of an expressed cellular sap and the water potentials of ice indicated almost ideal freezing behaviour and suggested equilibrium freezing. On the basis of the osmotic potentials of expressed cellular sap, the fractions of frozen cellular water which correspond to the measured water potentials of the frozen leaves could be determined (e.g. 74% at -3.0° C). The freezing points of leaves were found to be in the range between 0° C and -0.5° C, rendering evidence for freezing of almost pure water and thus confirming the conclusions drawn from the water-potential measurements. The leaves proved to be frost resistant down to temperatures between -5° C and -15° C, as depending on the species. They tolerated short supercooling periods which were necessary in order to start ice nucleation. Extracellular ice caps and ice crystals in the intercellular space were observed when cross sections of frozen leaves were investigated microscopically at subfreezing temperatures.Symbols T temperature - water potential Dedicated to Professor Dr. Hubert Ziegler on the occasion of his 60th birthday     

Effects of freezing to −196 °C and thawing on Setaria lutescens seeds

The effects of single and repeated freezing and thawing of Setaria lutescens seeds in liquid nitrogen were investigated. One freeze to ?196 °C followed by a slow thaw, increased seed germination from 40 to 70%, but additional freeze-thaw cycles reduced germination to 30%. Using a scanning electron microscope, evidence was produced that seed coat cracking did not cause either initial increased, or subsequent reduced germination. Observations with a transmission electron microscope revealed that disruption of the integrity of lipid bodies accompanied increased damage from repeated freezing at ?196 °C and thawing. Repeated freezing and thawing of seeds stored in liquid nitrogen should be done with care to avoid loss of the germplasm.     

Does freezing and dynamic flexing of frozen branches impact the cavitation resistance of Malus domestica and the Populus clone Walker?

Frost damage to the xylem conduits of trees is a phenomenon of eco-physiological importance. It is often documented in terms of the percentage loss of conductivity (PLC), an indicator of air filling of the conduits. However, trees that refill their conduits in spring could be impacted more by damage to the conduits that reduce cavitation resistance, making them more susceptible to future drought events. We investigated whether ice formation, dynamic flexing of frozen branches or freeze–thaw events could reduce the cavitation resistance (cause “frost fatigue”) in first-year shoots of apple (Malus domestica) and clonal hybrid cottonwood (Walker). Frost fatigue was measured in terms of P₅₀ (the negative xylem pressure required to cause a 50 % loss of conductivity). All treatment groups showed significant frost fatigue, with the exception of the pre-flushed, constantly frozen poplar branches. The P₅₀ following freeze treatments was approximately 50 % of the pre-freeze values. The effect tended to be greater in freeze–thawed branches. Dynamic bending of the branches had no effect on either PLC or P₅₀. In three out of four cases, there was a significant correlation between P₅₀ and PLC. Frost fatigue occurred in both apple and poplar, two unrelated species with different drought and frost tolerances, suggesting that it may be a widespread phenomenon that could impact the ecophysiology of temperate forests.     

Brown bear sperm double freezing: Effect of elapsed time and use of PureSperm® gradient between freeze–thaw cycles

The use of sexed spermatozoa has great potential to captive population management in endangered wildlife. The problem is that the sex-sorting facility is a long distance from the semen collection place and to overcome this difficulty two freeze–thaw cycles may be necessary. In this study, effects of refreezing on brown bear electroejaculated spermatozoa were analyzed. We carried out two experiments: (1) to assess the effects of the two freezing–thawing cycles on sperm quality and to analyze three different elapsed times between freezing–thawing cycles (30, 90 and 180 min), and (2) to analyze the use of PureSperm between freezing–thawing cycles to select a more motile and viable sperm subpopulation which better survived first freezing. The motility, viability and undamaged acrosomes were significantly reduced after the second thawing respect to first thawing into each elapsed time group, but the elapsed times did not significantly affect the viability and acrosome status although motility was damaged. Our results with the PureSperm gradient showed higher values of viability in freezability of select sample (pellet) respect to the rest of the groups and it also showed a significant decrease in the number of acrosome damaged. In summary, the double freezing of bear semen selected by gradient centrifugation is qualitatively efficient, and thus could be useful to carry out a sex-sorting of frozen–thawed bear spermatozoa before to send the cryopreserved sample to a biobank. Given the low recovery of spermatozoa after applying a selection gradient, further studies will be needed to increase the recovery rate without damaging of the cell quality.     

Do heat shock proteins provide protection against freezing?

Yeast cells were frozen by plunging directly into liquid nitrogen (LN2) after exposure at 43 degrees C. Both the cells frozen without prior exposure to heat shock and those treated with cycloheximide showed almost 100% loss of viability during freezing and thawing. Heat exposure prior to freezing and thawing significantly increased the cell viability. This increase in cell viability was associated with the induction of heat shock protein synthesis, which was detected by gel electrophoresis. This protein may act by stabilizing the macromolecules and by increasing the hydrophobic interactions.     

Does the cryogenic freezing process cause shorter telomeres?

We have observed evidence of increased telomere shortening in short-term T-lymphocyte cultures following freezing and thawing of the original inoculum obtained by ficoll-paque gradient centrifugation, compared to T-lymphocytes that were cultured immediately without freezing and thawing from the same blood sample from 3 female and 3 male adults. Because freezing may have similar effects on other cell types, and because telomere shortening may only manifest its effects after many years or decades, we suggest there is a pressing need for evaluation of the effects of freezing on any cells envisioned for clinical applications, including embryo implantation.