Cryobiology

The aim of this study was evaluation of ram semen freezability treated with different rate of α-tocopherol in non-breeding season. In this study, ejaculates were collected from four Merino rams (2-3 ages were used and belonging to the special sheep farm in Bademli, Burdur/Turkey). The ejaculates containing spermatozoa with >80% motility and concentrations higher than 1.5x109 spermatozoa/ml (normozoospermic) were mixed and used in the study. The mixed ejaculates were divided into five equal aliquots and samples were extended with tris base medium (tris, citric acid, glucose) containing 800 µM group, 400 µM group, 200 µM group, 100 µM group or 0 µM group (control) α-tocopherol and they were equilibrated to 4°C for 2 hours, frozen in mini straws then stored in liquid nitrogen. Straws were thawed at 37°C for 30s in water bath for plasma membrane acrosome integrity (PMAI %) and high mitochondrial membrane potential (HMMP %) with flow cytometric analyses. Flow cytometry analyses were performed with a Cytoflex Flow Cytometer (Beckman Coulter, Fullerton, CA, USA). Assessments of semen samples were made with a laser beam at 488 nm (50 mW laser output), with 525 ± 40, and 585 ± 42 and 610 ± 20 nm emission filters. Data were collected from 10.000 events. The highest PMAI (24.39±2.11 %) and HMMP (39.90±3.13 %) were in 100 µM compared to other groups (p<0.05). Also, the lowest PMAI and HMMP values were obtained in 800 µM and 400 µM groups respectively (p<0.05). In conclusion, 100 µM α-tocopherol should be supplemented to ram semen freezing extender for membrane & acrosome integrity and high mitochondrial membrane potential.

The aim of this study was evaluation of ram semen freezability treated with different rate of α-tocopherol in non-breeding season. In this study, ejaculates were collected from four Merino rams (2-3 ages were used and belonging to the special sheep farm in Bademli, Burdur/Turkey). The ejaculates containing spermatozoa with &gt;80% motility and concentrations higher than 1.5x109 spermatozoa/ml (normozoospermic) were mixed and used in the study. The mixed ejaculates were divided into five equal aliquots and samples were extended with tris base medium (tris, citric acid, glucose) containing 800 μM group, 400 μM group, 200 μM group, 100 μM group or 0 μM group (control) α-tocopherol and they were equilibrated to 4°C for 2 hours, frozen in mini straws then stored in liquid nitrogen. Straws were thawed at 37°C for 30s in water bath for plasma membrane acrosome integrity (PMAI %) and high mitochondrial membrane potential (HMMP %) with flow cytometric analyses. Flow cytometry analyses were p...

Cellular membranes are one of the primary sites of injury during freezing and thawing for cryopreservation of cells. Fourier transform infrared spectroscopy (FTIR) was used to monitor membrane phase behavior and ice formation during freezing of stallion sperm. At high subzero ice nucleation temperatures which result in cellular dehydration, membranes undergo a profound transition to a highly ordered gel phase. By contrast, low subzero nucleation temperatures, that are likely to result in intracellular ice formation, leave membrane lipids in a relatively hydrated fluid state. The extent of freezing-induced membrane dehydration was found to be dependent on the ice nucleation temperature, and showed Arrhenius behavior. The presence of glycerol did not prevent the freezing-induced membrane phase transition, but membrane dehydration occurred more gradual and over a wider temperature range. We describe a method to determine membrane hydraulic permeability parameters (E Lp , Lpg) at subzero temperatures from membrane phase behavior data. In order to do this, it was assumed that the measured freezinginduced shift in wavenumber position of the symmetric CH 2 stretching band arising from the lipid acyl chains is proportional to cellular dehydration. Membrane permeability parameters were also determined by analyzing the H 2 O-bending and -libration combination band, which yielded higher values for both E Lp and Lpg as compared to lipid band analysis. These differences likely reflect differences between transport of free and membrane-bound water. FTIR allows for direct assessment of membrane properties at subzero temperatures in intact cells. The derived biophysical membrane parameters are dependent on intrinsic cell properties as well as freezing extender composition.

Cnidarian primary cell cultures have a strong potential to become a universal tool to assess stress-response mechanisms at the cellular level. However, primary cell cultures are time-consuming regarding their establishment and maintenance. Cryopreservation is a commonly used approach to provide stable cell stocks for experiments, but it is yet to be established for Cnidarian cell cultures. The aim of this study was therefore to design a cryopreservation protocol for primary cell cultures of the Cnidarian Anemonia viridis, using dimethyl sulfoxide (DMSO) as a cryoprotectant, enriched or not with fetal bovine serum (FBS). We determined that DMSO 5% with 25% FBS was an efficient cryosolution, resulting in 70% of post-thaw cell survival. The success of this protocol was first confirmed by a constant post-thaw survival independently of the cell culture age (up to 45 days old) and the storage period (up to 87 days). Finally, cryopreserved cells displayed a long-term recovery with a maintenance of the primary cell culture parameters and cellular functions: formation of cell aggregates, high viability and constant cell growth, and unchanged intrinsic resistance to hyperthermal stress. These results will further bring new opportunities for the scientific community interested in molecular, cellular, and biochemical aspects of cnidarian biology.

Minimizing cell damage throughout the cryopreservation process is critical to enhance the overall outcome. Osmotic shock sustained during the loading and unloading of cryoprotectants (CPAs) is a major source of cell damage during the cryopreservation process. We introduce a microfluidic approach to minimize osmotic shock to cells during cryopreservation. This approach allows us to control the loading and unloading of CPAs in microfluidic channels using diffusion and laminar flow. We provide a theoretical explanation of how the microfluidic approach minimizes osmotic shock in comparison to conventional cryopreservation protocols via cell membrane transport modeling. Finally, we show that biological experiments are consistent with the proposed mathematical model. The results indicate that our novel microfluidic-based approach improves post-thaw cell survivability by up to 25% on average over conventional cryopreservation protocols. The method developed in this study provides a platform to cryopreserve cells with higher viability, functionality, and minimal inter-technician variability. This method introduces microfluidic technologies to the field of biopreservation, opening the door to future advancements at the interface of these fields.

keratinocyte osmotic response to alteration in composition of the bathing medium are recorded for subsequent digitization. Trials are conducted both with and without glycerol present in the control solutions. For each cell image a pseudovolume is calculated from its measured perimeter. The succession of these pseudovolumes is then inserted into a microcomputer based curve fitting program, implementing a network thermodynamic model of the cell, to evaluate the Kedem-Katchalsky coefficients of the membrane. (Sponsored by a grant from the Texas Advanced Technology Research Program.) 33. Measurement of Osmolic Properties of Monocytes on a Microscopic Diffusion Chumber.

The freeze tolerance and accumulation of cryoprotectants was investigated in three geographically different populations of the enchytraeid Enchytraeus albidus (Oligochaeta). E. albidus is widely distributed from the high Arctic to temperate Western Europe. Our results show that E. albidus is freeze tolerant, with freeze tolerance varying extensively between Greenlandic and European populations. Two populations from sub Arctic (Nuuk) and high Arctic Greenland (Zackenberg) survived freezing at À15 °C, whereas only 30% of a German population survived this temperature. When frozen, E. albidus responded by catabolising glycogen to glucose, which likely acted as a cryoprotectant. The average glucose concentrations were similar in the three populations when worms were frozen at À2 °C, approximately 50 lg glucose mg À1 tissue dry weight (DW). At À14 °C the glucose concentrations increased to between 110 and 170 lg mg À1 DW in worms from Greenland. The average glycogen content of worms from Zackenberg and Nuuk were about 300 lg mg À1 DW, but only 230 lg mg À1 DW in worms from Germany showing that not all glycogen was catabolised during the experiment. Nuclear magnetic resonance spectrometry (NMR) was used to screen for other putative cryoprotectants. Proline, glutamine and alanine were up regulated in frozen worms at À2 °C but only in relatively small concentrations suggesting that they were of little significance for freeze survival. The present study confirms earlier reports that freeze tolerant enchytraeids, like other freeze tolerant oligochaete earthworms, accumulate high concentrations of glucose as a primary cryoprotectant.

The effect of the low intensity centrifugation (300xg), as well as the breed variation were evaluated. To this, semen of 12 dogs of the Beagle (n = 3), Schnauzer (n = 3), Doberman (n = 3) and Boxer (n = 3) breeds, 3 ejaculates collected per individual, totaling 36 ejaculates were used. The direct microscopic evaluation of the motility parameters and the hypoosmotic and supravital staining tests were used. The individual means of sperm vigor and motility varied from 3.5 to 5.0 and from 63 to 90% respectively, while the breed means ranged from 4.4 to 4.9 and from 80 to 90%, respectively. No significant difference was observed in any of the parameters evaluated in fresh or centrifuged semen of dogs among the different breeds studied. Centrifugation of semen resulted in a reduction of about 13% in sperm vigor and 14% in motility and sperm index. The analysis of the parameters of viability and sperm membrane integrity shows a 38% and 14% reduction in the percentage of reactive cells to the hypoosmotic test and supravital staining respectively. These results allow us to conclude that the centrifugation protocol, even at low intensity, significantly reduces sperm motility, the membrane integrity and sperm viability, this effect being more clearly perceived through the hypoosmotic test.

Liquid nitrogen is the most common medium used by tissue banks for the storage of cryopreserved heart valves. This study evaluates the effect of the length of storage on human cryopreserved heart valves. Human tissues (14 aortic and 13 pulmonary) were frozen in a controlled-rate freezer (1 °C/min) and stored in the liquid phase of a nitrogen tank for 9.1 ± 1.6 years. The preservative solution was medium M199 containing 5% human serum albumin and 10% Me 2 SO. After thawing in a water bath at 42 °C, the cryoprotectant was removed. Then, fragments from vascular wall and leaflet were dissected. Explant cultures and histological studies were performed in order to assess cell viability and structural integrity. CD90 and CD31 expression was analysed in cultured cells using flow cytometry. Light microscopy, immunofluorescence staining and laser scanning confocal microscopy were used to evaluate cell viability and extracellular matrix components. Electron microscopy was used for ultrastructural study. Cell cultures could be obtained from all the specimens assayed. Cells grew from explants showing a fibroblastic phenotype. CD90 expression was common in cultured cells but a low percentage of cells expressed CD31. Histological results showed a good preservation estructure in both leaflets and vascular walls. Morphological features of cellular irreversible damage were very rare. No differences which could be due to length of allograft storage period were observed. We concluded that allografts stored in liquid nitrogen up to 13 years did not significantly undergo loss of cell viability other than that due to disinfection, freezing and thawing protocols.

Biobanking is done almost exclusively by cryopreservation, followed by maintenance of the samples under liquid nitrogen. Cryopreservation has satisfactory efficiency but it comes with a host of problems, including high costs, need for guaranteed continuous liquid nitrogen and power supply, dedicated staff and storage facilities, complicated transportation, risk of bacterial/fungal/viral contamination and high carbon footprint. The process is also highly species-specific and damaging to the cells. Because of these reasons, biobanking is largely restricted to the Western/Developed World and even there, mainly to relatively large and advanced centres. Like in many other walks of life, we can search Nature for better alternatives. Water content in most organisms is close to 90% yet long-term preservation strategies are more efficient when grounded in water exclusion via controlled drying rather than in water preservation via freezing. Nature also created an assortment of materials that protect organisms from damages during the drying and rehydration processes (e.g. disaccharides, LEA proteins). Once dry, and in the absence of water, metabolism and chemical reactions within the cells are brought to a halt so that these cells can survive extended periods of time and be resistant to extreme environmental conditions. Desiccation is experiencing increased interest in recent years with some exciting developments. Still, the cells normally "die" along the process. Working on spermatozoa and somatic cells from large mammals (cattle, sheep, pigs, humans), we try to understand what drying really does to the cells and what we can do to protect them. Work on this is in progress. Presented here are interim results related to DNA integrity in such desiccated cells. Once the desiccation process matures, it will facilitate long-term sample preservation at ambient temperatures, making banking drastically less costly and accessible to third-world countries, SMEs and may even be available for home storage.

Dissociated mantle cells from the gastropod mollusc Haliotis tuberculata were cultured after a freezing-thawing procedure using either 10% dimethyl sulfoxide (Me 2 SO) or 10% glycerol (Gly) as a cryoprotector. The survival rate of 2-day-old cultured cryopreserved cells after thawing, based on analysis of DNA and protein contents, was nearly 80% in comparison with 2-day-old cultured fresh cells. Cells thawed after cryopreservation exhibited the maintenance of all tested physiological activities. Metabolic activity (measured by the MTT test) and the activity of alkaline phosphatase (a plasma membrane-bound enzyme) were not decreased in comparison to those in cultured fresh cells. In addition, cryopreserved cultured cells maintained a physiological stimulation ability in response to treatment with growth factors. These results taken together represent one of the most convincing demonstrations of the survival and of the recovery of intact functional activities of molluscan cells after a freeze-thawing procedure. Our results suggest that in the future primary cultures of cryopreserved mantle cells will be able to be used for fundamental research, in toxicity tests, or in the field of biotechnology. Ó 2002 Elsevier Science (USA). All rights reserved.

parameters of the plant seeds kept in the cryodepository testify to the maintenance of their biological properties, above all their germination capacity which is 95-100%: the level of chromosomal aberrations is about 1-4%. At the same time, storage of analogous seeds from collection of AURIP using standard methods for 11-13 years of storage resulted in the decline of germination to 50-80%, and the level of chromosomal aberration increased to 6-8%: storage for 28-30 years under these conditions would cause the seeds to become fully nonviable. Plants grown from the seed kept in a depository do not differ from plants grown from fresh seed when assessed by biomorphological indexes. There is a decrease in the rate of protein synthesis to 60-70% and of cell division to 70-95%. There is a growth of 2-3 times in the activity of protective antioxidant processes and of 20-30% in the activity of DNA repair systems, resulting in an increase in the integral index of genomic stability of 1-2 times. Our preliminary results justify questions about building a specialized cryodepository in Yakutia, in the layer of permafrost using natural cold for the creation of an economic, very cost-effective and ecologically clean genetic bank for the storage of plant seeds, and subsequently of organs, embryos and tissues from animals. Such genetic banks are needed for the maintenance of genetic resources, not only of cultural plants and domestic animals but also for the maintenance of rare and vanishing forms of wild-life required for the maintenance of global biological variety.

Aim: To examine whether thermo-perfusion of the bile duct and duodenum may protect these organs during cryoablation of adjacent pancreatic tissue. Study design: Cryoablation of the pancreatic tissue, adjacent to the common bile duct and duodenum was performed in two groups of pigs. In the experimental group, the bile duct and duodenum were protected during the cryo-procedure by intraluminal perfusion of warm saline. In the control group, cryoablation was performed without thermo-protection. Results: All three animals in the control group developed duodenal perforation and abscesses and died within a week. All the pigs in the experimental group survived and on re-operation 14 days after the first procedure were found to have normal duodenum and bile duct adjacent to the cryoablated pancreatic tissue. Histological examinations confirmed these results. The present study confirms the feasibility and efficacy of thermo-protection of the duodenum and common bile duct during cryoablation of the head of the pancreas.

The possible roles of reduced glutathione (GSH) in chilling tolerance were studied in callus generated from a representative alpine plant, Chorispora bungeana Fisch. & C.A. Mey (C. bungeana). The callus grew well under low-temperature and chilling treatment led only to slight injury, as indicated by a low level of ion leakage (IL). Malondialdehyde measurements also were not elevated, however GSH was. Exogenously application of L-buthionine-(S R)-sulfoximine (BSO), an inhibitor of c-glutamylcysteine synthetase (c-ECS), arrested the GSH accumulation induced by chilling and resulted in a significant decrease in cell growth and an increase in IL and malondialdehyde. These results implied that C. bungeana is a plant with a strong low-temperature tolerance mechanism, and the tolerance of C. bungeana may be associated with GSH accumulation. Under chilling treatment, the proportion of unsaturated fatty acid in the plasma membrane (PM) increased significantly in callus of C. bungeana mainly due to increases in C18:2 and C18:3, the membrane fluidity (indicated by DPH fluorescent polarization) however was maintained. High PM H + -ATPase activities were also induced by chilling. Exogenously application of BSO blocked the effects of chilling treatments on the changes of fatty acids and PM H + -ATPase activities, reducing the PM membrane fluidity. On the other hand, simultaneous application of GSH and BSO to callus under chilling treatments reversed the effects of BSO on the changes of fatty acids, PM fluidity and PM H + -ATPase activities. These results suggested that GSH induced by low-temperature treatments may confer chilling tolerance to C. bungeana, probably by increasing unsaturated fatty acid compositions and maintaining PM fluidity and high enzymatic activity.

In this study, total glutathione content was determined in human spermatozoa before and after cryopreservation. Total GSH in fresh semen was 4.47 ± 0.46 nmol/10 8 cells. Following semen cryopreservation, GSH decreased to 1.62 ± 0.13 nmol/10 8 cells, a 64% reduction (p < 0.01). This decrease in GSH content was associated with a decrease in sperm progressive motility (68% of reduction, p < 0.01). Addition of 1 mM GSH to the freezing extender increased the percentage of total motility and sperm viability. It also modified the motility pattern measured by CASA with changes in the straight-line and average path velocities and wobble of the curvilinear trajectory. Addition of GSH to the freezing media reduced spermatozoa ROS levels and increased the level of sulfhydryl groups on membrane proteins. Nevertheless, no effect of GSH addition on lipid membrane disorder or chromatin condensation was detected. Addition of 1 or 5 mM GSH to the thawing media increased the percentage of motile and progressively motile spermatozoa, but no effect on viability was detected. In conclusion, the antioxidant defensive capacity of the GSH is severely altered by the freeze-thawing process. The addition of GSH to the freezing and thawing extender could be of partial and limited benefit in improving the function of frozen human spermatozoa.

This study investigated whether the activities of four antioxidant enzymes present in jackass seminal plasma (SP), superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX) and glutathione reductase (GSR), are related to the sperm ability to withstand cryopreservation. Eighteen ejaculates from 16 healthy jackasses were collected and split into two aliquots. The first one was centrifuged (3,000�g, 4 � C for 10 min) and used to determine the activities of these four enzymes in SP, whereas the other was diluted in a skim-milk extender and then cryopreserved. Assessment of sperm motility and membrane integrity was performed before and after cryopreservation. Based on the percentages of total motile and viable spermatozoa at post-thaw, samples were classified as good (GFE) or poor (PFE) freezability ejaculates through cluster analyses. Total and specific activities of SOD in seminal plasma were higher (P < 0.05) in GFE than in PFE, whereas no significant differences between GFE and PFE were observed regarding total and specific activities of CAT, GPX and GSR. However, post-thaw sperm parameters were positively correlated with total and specific activities of CAT and negatively correlated with those of GSR. In conclusion, determination of total and specific activities of SOD in the seminal plasma of a given jackass ejaculate may predict the sperm ability to withstand cryopreservation. In addition, our results warrant further research on addressing whether SOD activity in seminal plasma does not only allow predicting the sperm cryotolerance of a given ejaculate but also that of all ejaculates from a given jackass.

Almtraet-1. Hatchling snapping turtles (Chelydra serpentina) that were placed on platforms of dry styrofoam survived for 7 h without freezing at temperatures below-4°C. 2. Turtles that were confined to artificial nests in frozen soil were unable to withstand exposure to temperatures much lower than-2°C. 3. Animals that were held in dry surroundings and then cooled to-2°C froze rapidly when they were brought into contact with crystals of ice. 4. These observations indicate that turtles which die from cold exposure when they are forced to overwinter in nests in the field probably succumb to inoculative freezing caused by contact with ice in the soil and not to spontaneous nucleation caused by body fluids reaching their limit of supercooling.

P ainted turtles (Chrysemys picta) are common residents of shallow lakes and marshes across much of North America east of the Rocky Mountains-so common, in fact, that the species has become a "model organism" for studies on the ecology and evolution of chelonians (Wilbur and Morin 1988). The natural history of painted turtles differs from that of most other species in an important respect, however. Whereas neonates of other freshwater turtles usually emerge from their subterranean nest in late summer or autumn and move to a nearby marsh, lake, or stream to spend their first winter, hatchling painted turtles typically remain inside their shallow (8-14 cm) nest throughout their first winter and do not emerge above the ground until the following spring (Ernst et al. 1994). This behavior commonly causes neonatal painted turtles in northern regions-from Nebraska (Packard 1997, Packard et al. 1997a), northern Illinois (Weisrock and Janzen 1999), and New Jersey (DePari 1996) northward to the limit of distribution in southern Canada (Storey et al. 1988)-to be exposed during winter to ice and cold, with temperatures in some nests dipping below-10°C (Figure 1). Many of these hatchlings withstand such extremes and emerge from the nest when the soil finally thaws in the spring (Table 1).

The inherent leakiness of the tumor neovasculature allows for improved biodistribution of systemically administered nanomedicines. However, the high interstitial fluid pressure (IFP) created by these leaky tumor blood vessels may also prevent such constructs, as well as systemically administered chemotherapy or immunotherapy, from being maximally effective. One way to improve the efficacy of anti-cancer agents would be through the elimination of the high tumor IFP. CYT-6091, a nanomedicine comprised of tumor necrosis factor alpha (TNF) bound to PEGylated gold nanoparticles, has been shown to cause vascular leak in two preclinical tumor models. Associated with that vascular leak was a signficant reduction in the high tumor IFP in implanted tumors, and a 6-fold increase in paclitaxel accumulation inside naturally occurring tumors in genetically engineered mice. CYT-6091 has been successfully tested as a single agent in a phase I clinical trial in advanced-stage cancer patients. Success in a CYT-6091 plus chemotherapy phase II study will demonstrate that cancer patients should be treated with CYT-6091 prior to chemotherapy in order to reduce tumor size in situ, potentially leading to less complicated followon surgery, less time in hospital, improved patient outcomes, and ultimately reduced healthcare cost associated with treating patients with cancer.

Domestic violence a social evil against women and can be defined as "a pattern of behaviour in any relationship that is used to gain or maintain power and control over an intimate partner". It is a matter of serious public health concerns at all the places around the globe. This problem cannot be resolved without mutual effort of "law enforcement, social welfare and health care services". The problem resolution gained significance in India after domestic violence act in 2005. Main objective of the paper is to assess the occurrence of domestic violence against women, and to study the effects and causes of domestic violence in Indian society. The study also aims at finding out the socio-demographic factors in females suffering from domestic violence. Using the Research Methodology: A population based cross sectional study was carried out in the rural areas of Jaipur district for 4 months. The total population covered under this study was around 35 Km. Samples were selected using Simple random sampling method. The respondents selected for the study were interviewed using a predesigned structured questionnaire. Different frequency tables and graphs were used for the analysis of the study and Finding Indian rural women are still facing a very high percentage of domestic violence against them and hence the problem needs urgent and serious resolution which should be done by collaborative endeavours of society, people and government. Rural women are required to be empowered to stand against this social evil. Generating awareness, changing mind-set of people and society with the help of education and law enforcement can only brought real change in these cases.

The aim of the present study was to evaluate the effectiveness of human ovarian vitrification protocol followed with in vitro culture at the morphological and molecular levels. Ovarian tissues were obtained from 10 normal transsexual women and cut into small pieces and were divided into non-vitrified and vitrified groups and some of the tissues fragments in both groups were randomly cultured for two weeks. The morphological study using hematoxylin and eosin and Masson&#39;s trichrome staining was done. The analysis of mean follicular density, 17-β estradiol (E2) and anti mullerian hormone (AMH), and real-time RT-PCR was down for the evaluation of expression of genes related to folliculogenesis. Data were compared by paired-samples and independent-samples T test. Values of p&lt;0.05 were considered statistically significant. The proportion of normal follicles did not show significant difference between vitrified and non-vitrified groups before and after culture but these rates and th...

Tegenwoordig zijn veel landbouwbedrijven op zoek naar manieren om hun activiteiten te diversifieren, en in die context kan de teelt van rivierkreeft mogelijk een interessante en duurzame uitkomst bieden. Twee Belgische hogescholen – Katholieke Hogeschool Sint-Lieven (KAHO) en Hogeschool Universiteit Brussel (HUB) – onderzoeken of de productie van Europese rivierkreeft een leefbare activiteit kan zijn voor Belgische landbouwers.

Differential Scanning Calorimetry (DSC), a classic biophysical technique for characterizing thermal transitions in the triacylglycerol phase of dry seeds, has shown links to storage behavior. Though a powerful tool, DSC has some limitations. Fourier Transform Infrared (FT-IR) spectroscopy provides fast and valuable physicochemical information due to its sensitivity to hydrogen bonds, being an option for accurate analysis of changes in lipid structures. Lipid's absorptivity in the infrared region allows higher pathlengths than other methods. Tomato seeds deterioration for different varieties stored for 5-31 years at the COMAV Plant Germplasm Bank (Valencia, Spain) was studied using traditional (germination and tetrazolium test) and non-traditional (DSC and FT-IR) techniques, to rapidly identify the accessions physiological status. Samples exhibited an exothermic event (-110 to-57 ºC), attributed to lipid crystallization and at least two lipids melting peaks (-50 to 5 ºC). Melting peak temperatura differed among accessions whereas devitrification energy decreased with storage time, with exception of samples harvested in 1999, showing anomalous results. FT-IR spectra from whole seeds were processed by Principal Components Analysis (PCA), where two principal components explained 89.5% of the total variance. PC1 (71%) was positively correlated with the peak at 1600 cm-1 related to the Amide I zone, denoting any dissimilarities in protein structure, whereas PC2 was strongly related to lipid peaks. Also, to evaluate the degree of unsaturation in stored samples, specific FT-IR absorbance ratios were calculated, estimating iodine values: one of the accessions showed higher values (p<0.05). The relationship between seed storage time and lipid behavior, assessed by DSC and FT-IR, was the same that had been observed by germination and tetrazolium test. All methods employed allowed detecting differences among varieties or accessions of the same variety. The integrated approach of traditional and non-traditional methods helps understanding the total data set, allowing an accelerated seed quality assessment.

Introduction of urethral warmers to aid cryosurgery in the prostate has significantly reduced the incidence of urethral sloughing; however, the incidence rate still remains as high as 15%. Furthermore, urethral warmers have been associated with an increase of cancer recurrence rates. Here, we report results from our phantom-based investigation to determine the impact of a urethral warmer on temperature distributions around cryoneedles during cryosurgery. Cryoablation treatments were simulated in a tissue mimicking phantom containing a urethral warming catheter. Four different configurations of cryoneedles relative to urethral warming catheter were investigated. For each configuration, the freeze-thaw cycles were repeated with and without the urethral warming system activated. Temperature histories were recorded at various prearranged positions relative to the cryoneedles and urethral warming catheter. In all configurations, the urethral warming system was effective at maintaining sub-lethal temperatures at the simulated surface of the urethra. The warmer action, however, was additionally demonstrated to potentially negatively impact treatment lethality in the target zone by elevating minimal temperatures to sub-lethal levels. In all needle configurations, rates of freezing and thawing were not significantly affected by the use of the urethral warmer. The results indicate that the urethral warming system can protect urethral tissue during cryoablation therapy with cryoneedles placed as close as 5 mm to the surface of the urethra. Using a urethral warming system and placing multiple cryoneedles within 1 cm of each other delivers lethal cooling at least 5 mm from the urethral surface while sparing urethral tissue.

Experiments were carried out on the sperm cryopreservation of artificially induced eels. The effects of several extenders and two cryoprotectants on the motility of spermatozoa were investigated. The highest post-thaw motility was observed with the combination of Tanaka's extender and DMSO as cryoprotectant. Further dilution after thawing resulted in complete loss of motility in samples frozen in presence of DMSO while sperm frozen with methanol as cryoprotectant retained its motility after further dilution.

cryoprobe within 5 sec and achieved a tip temperature of <À150 C within 10 sec. The device repeatedly generated ice balls of 3 cm diameter in less than 2 minutes. Heat load tissue model studies revealed the generation of a full thickness (8 mm) 2-e10 cm long cryogenic lesions (probe freeze zone dependent) within 1 min, achieving an opposite surface temperature of <-<À100 C. Preliminary animal studies demonstrated the delivery of an ablative cryogenic "dose", producing a contiguous transmural linear lesion in <90 seconds. These studies demonstrate that the SCN technology provides for rapid, effective, controllable freezing of targeted tissue. The ablative power, speed and directional freeze characteristics of the SCN system also offers the potential of improved safety via a reduction in procedural time compared to current cryoablation devices. Further, these technological developments may open new avenues for the application of cryo to treat other cardiac arrhythmogenic disorders including ventricular tachycardia.

Rabbit corneas were frozen and thawed by three methods and compared by full thickness transplantation as well as specular microscopy, histology, and transmission electron microscopy. Two of the methods used a recently described technique, in which the excised cornea was immersed in a potassium-rich buffered solution containing the cryoprotectant dimethyl sulphoxide (Me2SO, 2 mol/l). This solution was designed to restrict the loss of intracellular potassium and to prevent cell swelling at low temperatures. In one group the corneas were frozen and thawed surrounded by 5 ml of medium, while in the second group corneas were drained of excess fluid and frozen in air. The third group consisted of corneas cryopreserved by Capella and colleagues' method. All the cryopreserved corneas were damaged, but those that had been frozen in air after exposure to the new medium showed better structure and function than corneas frozen by either of the other two techniques.

The commercial and clinical development of cellular therapy products will invariably require cryopreservation and frozen storage of cellular starting materials, intermediates and/or final product. Optimising cryopreservation is as important as optimisation of the cell culture process in obtaining maximum yield and a consistent end-product. Suboptimal cryopreservation can lead not only to batch-to-batch variation, lowered cellular functionality and reduced cell yield, but also to the potential selection of subpopulations with genetic or epigenetic characteristics divergent from the original cell line. Regulatory requirements also impact on cryopreservation as these will require a robust and reproducible approach to the freezing, storage and thawing of the product. This requires attention to all aspects of the application of low temperatures: from the choice of freezing container and cryoprotectant, the cooling rate employed and its mode of delivery, the correct handling of the frozen material during storage and transportation, to the eventual thawing of the product by the end-user. Each of these influences all of the others to a greater or lesser extent and none should be ignored. This paper seeks to provide practical insights and alternative solutions to the technical challenges faced during cryopreservation of cells for use in cellular therapies.

Lake Vostok, the 7 th largest (by volume) and 4 th deepest lake on Earth, is covered by more than 3,700 m of ice, making it the largest subglacial lake known. The combination of cold, heat (from possible hydrothermal activity), pressure (from the overriding glacier), limited nutrients and complete darkness presents extreme challenges to life. Here, we report metagenomic/metatranscriptomic sequence analyses from four accretion ice sections from the Vostok 5G ice core. Two sections accreted in the vicinity of an embayment on the southwestern end of the lake, and the other two represented part of the southern main basin. We obtained 3,507 unique gene sequences from concentrates of 500 ml of 0.22 mm-filtered accretion ice meltwater. Taxonomic classifications (to genus and/or species) were possible for 1,623 of the sequences. Species determinations in combination with mRNA gene sequence results allowed deduction of the metabolic pathways represented in the accretion ice and, by extension, in the lake. Approximately 94% of the sequences were from Bacteria and 6% were from Eukarya. Only two sequences were from Archaea. In general, the taxa were similar to organisms previously described from lakes, brackish water, marine environments, soil, glaciers, ice, lake sediments, deep-sea sediments, deep-sea thermal vents, animals and plants. Sequences from aerobic, anaerobic, psychrophilic, thermophilic, halophilic, alkaliphilic, acidophilic, desiccation-resistant, autotrophic and heterotrophic organisms were present, including a number from multicellular eukaryotes.

In vitro follicle growth is a promising strategy for female fertility preservation. This study was conducted to compare the development of ovine follicles either isolated or in the context of ovarian cortical pieces after short term (8 days) three-dimensional culture in fresh and vitrified samples. Four different experiments were conducted; I) culture of ovarian cortical pieces encapsulated in 0.5% and 1% alginate and without alginate encapsulation (CP-0.5%, CP-1% and CP, respectively), II) culture of isolated primordial and primary follicles encapsulated in 1% and 2% alginate (IF-1% and IF-2%, respectively), III) culture of fresh and vitrified-warmed cortical pieces (F-CP and Vit-CP, respectively), and IV) culture of fresh and vitrified-warmed encapsulated isolated follicles (F-IF and Vit-IF, respectively). The number of secondary follicles after culture was negatively influenced by encapsulation of ovarian cortical pieces (6.3 ± 3.3 and 10.6 ± 0.9 vs 21.5 ± 2.3 in CP-0.5% and CP-1...

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Sperm cryopreservation is challenging, often resulting in irreversible damage to spermatozoa, as indicated by decreased motility, viability, and/or acrosomal integrity. Developing cryopreservation protocols for gametes of endangered species compounds the complexity of technique optimization; samples are difficult to obtain and numbers are limited. Cryopreservation of sperm collected from the critically endangered addra gazelle (Nanger dama ruficollis), a member of the Bovidae family, resulted in significant loss of motility, which was prevented by pretreatment with cholesterol-loaded cyclodextrin (CLC). This study investigated the proteome of sperm (fresh and cryopreserved), processed in the absence and presence of 0.5 mg/ml CLC in the addra gazelle. The proteome of Bos taurus, the closest domestic relative, was used as a reference. Mass spectrometry analysis of the addra gazelle sperm proteome revealed 287 proteins. The concentrations of 85 proteins differed between fresh and frozen/thawed samples; nearly all were decreased. Most were associated with metabolic processes, specifically glycolysis, which may explain the decrease in post-thaw motility observed in this species. CLC pretreatment partially prevented the loss of various proteins involved in metabolism including CAPZB (gene = CAPZB), HS90A (gene = HSP90AA1), and PGAM2 (gene = PGAM2). To our knowledge, this is the first study to evaluate the proteome of any wild bovids' sperm, and the first to compare protein levels in sperm pretreated with CLC.

German translation of Kowal, E. and Radin, J. (2015) Indigenous biospecimen collections and the cryopolitics of frozen life. Journal of Sociology 51(1): 63-80.

Published in Thomas Lemke, Elena Beregov & Veit Braun (eds.) Critical Temperature Studies: Konturen eines Forschungsprogramms. Frankfurt/New York: Campus Vorlag, pp263-288.

improves freezability. This study was designed to elucidate whether the inclusion of HT in the cryopreservation protocol can change the ejaculate freezability. Twenty-seven sperm-rich fractions were collected. Each sample was divided into two aliquots and extended 3-fold with Beltsville Thawing Solution. One aliquot was maintained at 17ºC/24 hours (HT24), the other was cryopreserved immediately (HT0). After, samples were centrifuged (2100xg/3min), sperm pellets were extended in cryopreservation medium without cryoprotectant. After cooling to 5 C/110 min, samples were re-diluted with cryopreservation medium (6% glycerol and 6% methylformamide) to 1x10 9 sperm/mL. Extended sperm were packed into 0.5mL straws, placed 3cm above liquid nitrogen level for 15min, then plunged into liquid nitrogen. Two straws per sample were thawed (37 C/ 30sec) and then, extended (TALP) to evaluation by the CASA system (TM e total motility) and also, by flow cytometry to assess the integrity of plasma and acrosome membrane (PAMI). Data were analyzed by PROC MIXED on SAS Studio®, means were compared using the Tukey Test. The ejaculates were rated as good (GEF) or poor (PEF) ejaculate freezability based on the following criteria of the HT24 samples: PAMI, GEF > 30 PEF; TM, GEF > 25 PEF. An interaction (p<0.05) between HT and freezability was found for all variables. In the case of GEF and PEF, respectively, we recorded the following parameters (p<0.05): TM (HT0: 12.82 ± 3.12 a , 12.82 ± 3.12 a ; HT24: 33.32 ± 2.41 a , 16.86 ± 2.01 b ) and PAMI (HT0: 17.42 ± 4.99 a , 11.61 ± 1.35 a ; HT24: 40.12 ± 3.35 a , 21.77 ± 2.93 b ). Thus, the freezability potential is associated with the incubation of sperm with own seminal plasma prior to freeze.