Bentonite is to be used as a sealing material for long-term storage of radioactive waste. During permafrost periods the buffer may freeze, causing the following: montmorillonite dehydration, ice formation, and pressure build-up that may fracture the surrounding rock. No previous study has been done on freezing of bentonite in saline water. Using small and wide angle X-ray scattering, the present study aimed to increase understanding of the combined impact of salt and temperature on the hydration (swelling) of Wyoming montmorillonite. The basal spacing of the Na-montmorillonite was very dependent on the water content, while this was not the case for the Ca-montmorillonite (after reaching 19 Å). The basal spacing of the free-swelling Na-montmorillonite (34–280 Å) was estimated successfully using simple calculations. During freezing of Na-montmorillonite in NaCl solution, both ice and hydrohalite formed (at -50 and -100ºC). At starting concentrations ≥ 1.5 M the basal spacing was not affected by freezing. During freezing of Ca-montmorillonite in CaCl2 solution, ice formed; antarcticite formed only sporadically. The basal spacing of the Ca-montmorillonite at high NaCl concentrations (>1 M) was greater at -50 and -100ºC (18 Å) than at 20ºC (16 Å). The opposite was observed at low concentrations. This change was attributed to small amounts of salts introduced into the montmorillonite interlayer, hence changing the interlayer water properties. The montmorillonite hydration was also temperature dependent; decreasing temperature increased the hydration (as long as no ice was formed) and increasing the temperature decreased the hydration. This was attributed to the temperature impact on the entropy of the hydration reaction. This observation was also reproduced in an experiment up to 90ºC. A small amount of salt in the groundwater was noted to reduce significantly the potential problem of ice formation in bentonite sealings.

Nematode spicules vary in shape and size even between closely related species and, therefore, constitute key characters in nematode taxonomy for distinguishing between species. Spicules are seldom measured on fresh specimens, but rather at some time after extraction from culled hosts and after a period of preservation of the worms in chemical fixatives or by freezing. We carried out two experiments to assess the effects of freezing in Hanks’ balanced salt solution, 70% or 80% ethanol and 10% formalin (both of the latter at room temperature and after storage at −80°C) on spicule length of Heligmosomoides bakeri at two time intervals after extraction from mice (Experiment 1, one and four weeks; Experiment 2, one and four months). In Experiment 1, no significant differences were detected, although there was some variation between treatments and over time. In Experiment 2, spicule length varied significantly between treatments and over time, the greatest shrinkage being in 80% ethanol and the least in 10% formalin. However, overall variation in spicule length was very limited, accounting for no more than 5.03% change in length over time and 4.95% between treatments at any of the periods of assessment. Therefore, while whole nematodes can shrivel and shrink in preservatives, making many measurements unreliable, our data indicated that spicule lengths are very little changed by preservation techniques over time, and so spicule length remains as a reliable taxonomic character.

Paramos are high-elevation tropical Andean ecosystems above the tree line that display variable temperature and frequent freezing spells. Because a significant anuran community lives in this environment, physiological protection against freezing must characterise individuals in this community. Antifreeze protection has been studied in amphibians from other communities, and it is likely that Paramo anurans rely on the same underlying molecules that convey such protection to Nearctic species. However, given the pervasive presence of freezing spells in the Paramos year-round, the processes of activating protection mechanisms may differ from that of seasonal counterparts. Accordingly, this study investigated cryoprotection strategies in high-elevation tropical frogs, using as a model the terrestrial and nocturnal genus Pristimantis, specifically P. bogotensis, P. elegans and P. nervicus from Paramos, and the warm ecosystem counterparts P. insignitus, P. megalops and P. sanctaemartae. We focused on freeze tolerance and its relationship with glucose accumulation and ice formation. Under field conditions, the highest elevation P. nervicus exhibited higher glucose concentration at dawn compared to noon (1.7 ± 0.6 mmol/L versus 3.5 ± 1.32 mmol/L). Under experimental thermal freeze exposure for 2 hours between −2 and −4 ºC, the glucose concentration of the three Paramo species increased but physiological diversity was evident (P. nervicus 126%; P. bogotensis 100%; and P. elegans 55%). During this test, body ice formation was assessed calorimetrically. The species with the highest body ice formation was P. bogotensis (17% ± 5.37; maximum value: 63%; n = 8), followed by P. nervicus (5% ± 3.27; maximum value: 11%; n = 5) and P. elegans (0.34% ± 0.09; maximum value: 1%; n = 4). The study shows physiological diversity both within a genus and across the amphibian community around the freezing contour. Overall, Paramo species differ in freezing physiology from their low-elevation counterparts. Thus, climate shifts increasing freezing spells may affect the structure of communities in this zone.

This article documents how members of the European Economic Community and members of the Arab League negotiated a draft ‘mega-regional’ investment protection treaty from 1976 to the late 1980s—the first of its kind. The negotiations produced a full draft treaty and came tantalisingly close to completion but ultimately ran into the political sands. Had it been concluded, the Convention would have been the most significant investment protection treaty ever negotiated at the time, and one of the most significant to this day. Negotiations were conducted within the cloak of diplomatic confidentiality, however, so the effort has remained unknown to even specialised scholars and practitioners to this day.

A major drawback of avocado (Persea americana Mill.) is susceptibility to frosts, which reduces yields and limits its geographic distribution and market growth. Whereas the frost-susceptible cultivar ‘Hass’ leads the global avocado market, cv. ‘Ettinger’, although commercially less important, is considered frost-tolerant. The mechanism behind the greater frost tolerance of ‘Ettinger’ has not yet been elucidated; therefore, the aim of the current study was to evaluate the differences between the frost responses of the two cultivars. The results showed that detached ‘Ettinger’ branches had greater tolerance than ‘Hass’ to controlled frost stress. Tissue browning caused by methyl viologen oxidative cell damage, superoxide accumulation in leaf discs following wounding and browning of cut surfaces in branches were much lower in ‘Ettinger’ than in ‘Hass’, suggesting greater antioxidant activity (AA) in the former. In leaf extracts, AA was significantly higher in ‘Ettinger’ than in ‘Hass’, but osmolarity was similar in the two cultivars. Total phenolics content was significantly higher in ‘Ettinger’ but addition of a protein mask did not significantly reduce AA in either cultivar. Interestingly, following the freezing treatment, AA increased in ‘Ettinger’ and remained almost unchanged in ‘Hass’, while osmolarity was unaffected in either cultivar. These results suggest that the greater frost-tolerance of ‘Ettinger’ than ‘Hass’ is due largely to its greater AA, which springs mainly from a non-enzymatic source, i.e. accumulation of phenolic compounds. Based on the current study, future applications may be developed to minimize frost damage in avocado orchards.

Laboratory and field experiments were conducted to describe the root system of honeyvine milkweed [Cynanchum laeve (Michx.) Pers.], and to study factors affecting the growth of plants from root fragments of shallow, ascending roots. The root system of a typical plant was composed of lateral roots radiating from a sparsely-branched, vertical taproot. Laterals tended to concentrate in a boundary area between a loamy surface soil and a zone of clay accumulation, this boundary area being below the zone of cultivation. Vertical taproots were found to a depth of 2 m, with sections collected at this and shallower depths demonstrating the ability to produce aerial shoots. In laboratory studies, root sections were killed either by freezing for 2 h or by drying at either 20 or 30 C for 24 h. The optimum temperature for shoot development from root sections was between 20 and 30 C. Shoot emergence and growth from buried root sections were generally unaffected by either depth of planting or root length. As many as 45 daughter shoots were produced from a single plant originating from seed, and 27 daughter shoots were developed from a root section 131 days after planting. The maximum distance daughter shoots were observed from original plants 131 days after planting was 111 cm.

Cognitive loading aggravates the freezing of gait (FoG), which is observed in approximately 50% of patients with Parkinson’s disease (PD) in the advanced stages. To investigate whether a specific pattern of executive deficits, that is, attentional set-shifting and/or inhibitory control, are associated with FoG in PD, 30 PD patients with FoG (PD-FoG+) and 36 PD patients without FoG (PD-FoG−) and 22 control healthy subjects were examined with a comprehensive neuropsychological battery. Intra-Extra Dimensional Set shifting Test (IED) and Stop Signal Task (SST), selected from the Cambridge Automated Neuropsychological Battery (CANTAB battery), were administered to analyze set-shifting and motor inhibition, respectively. The IED task was significantly sensitive for differentiating between PD-FoG+ and PD-FoG− groups (p<.01), as well Adenbrook’s clock drawing task (p=.033). By contrast, no differences emerged on any aspect of the SST task and other cognitive tasks. The attrition rate during the IED task showed that the problem in the PD-FoG+ group appeared at the pre-ID level, on the discrimination-learning set; the 32% PD-FoG+ subjects did not achieve the ID level of the task in comparison to negligible 4% of the PD-FoG− patients (p=.011). The logistic regression analysis, indicated the higher the IED stage successfully completed, the less likely presence of FoG in PD subjects. These results demonstrate that the complex cognitive–motor interplay might be responsible for FoG in PD and have had real life implication for the patients. (JINS, 2014, 20, 1–8)

We observed ice formation and water column attributes in four shallow Antarctic ponds between January and 7 April 2008. During that time ponds went from ice-free to > 80 cm thick ice, near-freshwater to hypersaline, well-lit to near darkness and temperatures fell to below zero. Here we examine shifts in biological activity that accompanied these changes. During February, freeze-concentration and ongoing photosynthesis increased dissolved oxygen concentration to up to 100 mg l-1, with a near-equivalent decrease in dissolved inorganic carbon and a pH rise. Benthic photosynthesis was responsible for 99% of estimated biological oxygen production. Net oxygen accumulation ceased in late February, pH began to fall and inorganic carbon to increase, but the pool of dissolved oxygen was depleted only slowly. Anoxia had been attained in only one pond by April and there was little accumulation of indicators of anaerobic activity. The nitrogen and phosphorus balances of the ponds were dominated by organic forms, which, like DOC and CDOM, behaved conservatively. Conversely, inorganic nitrogen and phosphorus uptake was evident throughout the study period, at a molar ratio of 16N:1P in two of three ponds, consistent with uptake into biological material. We found no coupling between N and P uptake and photosynthesis.

The effect of freezing on the properties of a raw ewes’-milk semi-soft cheese (Serpa cheese) was studied using small amplitude oscillatory (SAOS) and texture measurements, colour and chemical parameters. The freezing was introduced at three different stages of the ripening process (28, 35 and 42 days), and the cheeses were maintained frozen for 12 months. Cheeses were submitted to a slow or fast freezing method, and to different storage temperatures: −10 and −20°C (three replicates for each set conditions). Chemical data showed that only the proteolysis indicators exhibited differences between frozen and non-frozen samples; frozen samples showed higher values of NPN than the non-frozen samples, indicating that the freezing process did not prevent the secondary proteolysis of cheese. Frozen samples showed a significantly (P<0·05) stronger structure than the non-frozen, as indicated by hardness. However, the differences between the frozen and non-frozen samples were not significantly for storage modulus (G′1Hz) and loss tangent (tan δ1Hz) (P>0·05). Freezing affected mainly colour parameters: frozen samples were more luminous, and more yellow-green. The results allowed us to conclude that the damages caused by freezing to cheese properties could be minimized if this type of storage is introduced at the end of ripening (42 d) using a freezing temperature of −20°C.

Up to half of quarter milk samples submitted for mastitis diagnosis are culture-negative results or lead to identification of coagulase-negative staphylococci or Corynebacterium bovis in conventional culturing, the so-called minor pathogens. The interpretation and usefulness of these results in terms of udder and animal health management is limited, even though the amount of resources spent is relatively high. This work aimed to test two methods of analysis of milk samples with the goal of increasing detection of intramammary pathogens. In the first study, 783 milk samples were processed in duplicate: before and after freezing at −20°C for 24 h, using standard bacteriological techniques. There was a significant difference between the two methods with samples frozen for 24 h yielding significantly fewer Gram-positive catalase-positive cocci, Gram-negative bacilli, Gram-positive bacilli and significantly more samples leading to no growth, than samples before freezing. The number of samples yielding Gram-positive catalase-negative cocci was not significantly affected by freezing. In the second study, a real-time PCR-based test was performed on milk samples with an individual quarter somatic cell count above 500 000 cells/ml that were either negative (n=51 samples) or that led to the isolation of minor pathogens in culturing: Corynebacterium bovis (n=79 samples) or non-aureus staphylococci (NAS, n=32). A mastitis pathogen, beyond the result obtained with standard bacteriology, was detected on 47% of the no-growth samples, on 35% of the samples from which C. bovis had been isolated and on 25% of the samples from which NAS had been isolated. The most commonly detected major pathogen was Escherichia coli, followed by Streptococcus uberis, Arcanobacterium pyogenes/Peptoniphilus indolicus and Streptococcus dysgalactiae. These results suggest that simply freezing milk samples for 24 h does not increase the detection of intramammary bacteria in milk samples and therefore should not be recommended. However, use of the real-time PCR-based test may be useful in diagnosing intramammary infections when milk samples with high somatic cell counts are culture-negative or when culturing results in the detection of minor pathogens.

With the development of embryo technologies, such as in vitro fertilization, cloning and transgenesis, cryopreservation of mammalian gametes and embryos has acquired a particular interest. Despite a certain success, various cryopreservation techniques often cause significant morphological and biochemical alterations, which lead to the disruption of cell organelles, cytoskeleton damages, cell death and loss of embryo viability. Ultrastructural studies confirm high sensitivity of the cell membrane and organelle membrane to freezing and thawing. It was found that many substances with low molecular weights have a protective action against cold-induced damage. In this concern, an anti-freeze protein (AFP) and anti-freeze glycoproteins (AFGPs), which occur at extremely high concentrations in fish that live in Arctic waters and protect them against freezing, may be of potential interest for cryostorage of animal embryos at ultra-low temperatures. This mini-review briefly describes several models of AFP/AFGP action to preserve cells against chilling-induced damages and indicates several ways to improve post-thaw developmental potential of the embryo.

Low temperatures affect the rate of growth, development and metabolism of parasites and when temperatures fall below 0°C may expose the parasite to the potentially lethal risk of freezing. Some parasites have mechanisms, such as diapause, which synchronise their life cycle with favourable seasons and the availability of hosts. Parasites of endothermic hosts are protected from low temperatures by the thermoregulatory abilities of their host. Free-living and off-host stages, however, may be exposed to subzero temperatures and both freezing-tolerant and freeze-avoiding strategies of cold hardiness are found. Parasites of ectothermic hosts may be exposed to subzero temperatures within their hosts. They can rely on the cold tolerance adaptations of their host or they may develop their own mechanisms. Exposure to low temperatures may occur within the carcass of the host and this may be of epidemiological significance if the parasite can be transmitted via the consumption of the carcass.

The identification of early changes in the sperm plasmalemma is currently a factor in the improvement of freezing protocols. We analysed the presence of active caspases in freeze–thawed (FT) dog spermatozoa, and evaluated straws from eight dogs using flow cytometry and fluorescence microscopy with fluorescein isothyocyanate–Val–Ala–Asp–fluoromethylketone (FITC–VAD–fmk) combined with ethidum homodimers. Apoptotic-like changes were evaluated using the YO–PRO-1/ethidium homodimer combination, and changes in mitochondrial membrane potential were monitored with JC-1. Sperm motility post-thaw was evaluated using a CASA system. FITC–VAD–fmk stained sperm cells in situ and the subcellular labelling pattern was consistent with known localization of caspases. On average, a high proportion of FT canine sperm showed caspase activity, ranging from 30.2 to 70.7% of the live sperm compared with 7.3 to 24.0% in dead spermatozoa. This observed differentiation between caspase activity in dead and live spermatozoa may be a simple method to disclose subtle differences in sperm quality, since this staining allowed us to find statistically significant differences among dogs. Notably, the sperm sample with overall better results in all sperm parameters studied after thawing had a lower percentage of active caspases in both dead and live spermatozoa.

Hyaluronan (HA) is glycosaminoglycan that is present from the start of embryonic development and its role and concentration increases with embryo development. The objective of this study was to evaluate if the presence of HA in TCM-199 culture medium had an effect on the development and quality of bovine embryos. There was no effect of HA on the total number of zygotes developing to blastocysts on day 7, however more expanded and hatched blastocyst stages were observed on days 8 and 9 in the group supplemented with HA (p < 0.05). Following freeze/thawing, significantly more (p < 0.05) embryos cultured in medium supplemented with HA hatched than those cultured in TCM-199 alone or those with BSA. Medium supplemented with HA and BSA significantly increased the level of expression of glucose metabolism Glut-1 gene and embryo compaction Cx43 gene (p < 0.05), and had no effect on Glut-5 and IGF-II expression. In addition, HA presence in culture decreased the level of expression of apoptosis Bax and oxidative stress SOX genes (p < 0.05). There was significant difference in total number of nuclei between TCM-199 medium only and the remaining media containing BSA or HA plus BSA, between which there was no difference. In summary, our results indicate that the addition of high molecular weight HA to TCM-199 medium that contains BSA on day 4 of culture improved embryo development to hatching and hatched blastocysts and the quality of produced embryos, which were superior to embryos cultured without HA addition.

Semen cryopreservation in domestic birds has been studied extensively in the past fifty years. However, efficient methods to freeze semen of chicken of different breeds have emerged only in the last decade of the 20th century. Methods using the cryoprotectants glycerol, dimethyl acetamide and dimethyl sulfoxide, slow or rapid freezing-thawing procedures, pellets or vials or straws packaging have been developed. Other methods have been studied in turkeys, guinea fowl, ducks, ganders and some wild species. They are often less successful than chicken freezing methods with wide intra-species variability.

Reliable predictors of suitability of semen for cryopreservation are also needed to improve freezing methods and to optimize the management of frozen semen. The quality of whole fresh semen measured by classical morphologic, metabolic and mobility tests is an indicator. More specialized tests such as membrane fluidity of fresh spermatozoa are indicative of the freezing ability of the gametes.

The main use of semen cryopreservation in birds is the ex situ management of genetic resources in the context of dramatic decrease in avian genetic biodiversity. This is achieved through germplasm cryobanks. There are three main national avian germplasm cryobank programmes in 2006, operating in North America, The Netherlands and France. They mainly include semen and blood samples issued from Public Research Lines, indigenous breeds and individual specific genotypes.

The future need for semen cryopreservation programmes will focus mainly on three objectives 1) improvement of predictors of male suitability for semen freezing, 2) emergence of standardized methods of semen freezing in species other than chicken and 3) increasing development of avian cryobanks.

Kefir is an acidic, mildly alcoholic dairy beverage produced by the fermentation of milk with a grain-like starter culture (Koroleva, 1988). These grains usually contain a relatively stable and specific balance of microbes that exist in a complex symbiotic relationship (Obermann & Libudzisz, 1998; Witthuhn et al. 2004). The different groups of microbes present in the grains are active at different stages of the fermentation (Koroleva, 1982). The lactococci, including Lactococcus lactis subsp. lactis, Lc. lactis subsp. cremoris and Lc. lactis subsp. diacetilactis provide rapid acid development during the first hours of the fermentation (Litopoulou-Tzanetaki & Tzanetakis, 2000). As the acidity of the milk increases it provides favourable conditions for the growth of the lactobacilli (Rea et al. 1996). The yeasts, acetic acid bacteria and the aroma-producing microbes, mainly leuconostocs, have a much slower growth rate than the lactic acid producers, resulting in the slow production of the aroma compounds and the gradual increase in the concentration of these substances in the later stages of the fermentation (Koroleva, 1982).

Human faecal waste has been discarded at inland Antarctic sites for over 100 years, but little is known about the long-term survival of faecal microorganisms in the Antarctic terrestrial environment or the environmental impact. This study identified viable faecal microorganisms in 30–40 year old human faeces sampled from the waste dump at Fossil Bluff Field Station, Alexander Island, Antarctic Peninsula. Viable aerobic and anaerobic bacteria were predominantly spore-forming varieties (Bacillus and Clostridium spp.). Faecal coliform bacteria were not detected, indicating that they are less able to survive Antarctic environmental conditions than spore-forming bacteria. In recent years, regional warming around the Antarctic Peninsula has caused a decrease in permanent snow cover around nunataks and coastal regions. As a result, previously buried toilet pits, depots and food dumps are now melting out and Antarctic Treaty Parties face the legacy of waste dumped in the Antarctic terrestrial environment by earlier expeditions. Previous faecal waste disposal on land may now start to produce detectable environmental pollution as well as potential health and scientific problems.