The structure and long-term dynamics of the communities of Arctica islandica (Bivalavia) in Rugozerskaya Guba of the White Sea

The age structure of a dense settlement of Arctica islandica (Bivalvia) near Vysokii Island (Rugozerskaya Guba (bay)) of the White Sea is described. The data collected by trawling in 2003, 2006–2007 and grab sampling in 2009–2011 are summarized. The A. islandica community in the Rugozerskaya Guba was first described in 1953–1959, but to date, no reliable quantitative estimates have been obtained and the community’s size structure was not studied. The size structure of this community was stable over the last eight years; the main part of the community was represented by individuals with a body weight of 3–10 g. It was revealed that the distribution of different A. islandica classes depends on the depth. The majority of the largest individuals were found in the shallowest parts of the study area (9–11 m); middle-size individuals were registered at depths of 9–14 m; and the smallest ones, at depths of 12–17 m.     

Long-Time Cooling before Cryopreservation Decreased Translocation of Phosphatidylserine (Ptd-L-Ser) in Human Ovarian Tissue

Objectives

To translocation (externalization) of phosphatidylserine lead at least the five negative effects observed during cells cryopreservation: hypoxia, increasing of intracellular Ca²⁺, osmotic disruption of cellular membranes, generation of reactive oxygen species (ROS) and lipid peroxidation. The aim of this study was to test the intensiveness of the phosphatidylserine translocation immediately after thawing and after 45 d xenografting of human ovarian tissue, which was either frozen just after operative removal from patient or cooled before cryopreservation to 5°C for 24 h and then frozen.

Materials and Methods

Ovarian fragments from twelve patients were divided into small pieces in form of cortex with medulla, and randomly divided into the following four groups. Pieces of Group 1 (n=30) were frozen immediately after operation, thawed and just after thawing their quality was analyzed. Group 2 pieces (n=30) after operation were cooled to 5°C for 24 h, then frozen after 24 h pre-cooling to 5°C, thawed and just after thawing their quality was analyzed. Group 3 pieces (n=30) were frozen immediately after operation without pre-cooling, thawed, transplanted to SCID mice and then, after 45 d of culture their quality was analyzed. Group 4 pieces (n=30) were frozen after 24 h pre-cooling to 5°C, thawed, transplanted to SCID mice and then, after 45 d their quality was analyzed. The effectiveness of the pre-freezing cooling of tissuewas evaluated by the development of follicles (histology) and by intensiveness of translocation of phosphatidylserine (FACS with FITC-Annexin V and Propidium Iodide).

Results

For groups 1, 2, 3 and 4 the mean densities of follicles per 1 mm³ was 19.0, 20.2, 12.9, and 12.2, respectively (P_{1-2, 3-4} >0.1). For these groups, 99%, 98%, 88% and 90% preantral follicles, respectively were morphologically normal (P_{1-2, 3-4} >0.1). The FACS analysis showed significantly decreased intensiveness of translocation of phosphatidylserine after pre-cooling of frozen tissue (46.3% and 33.6% in Groups 2 and 4, respectively), in contrast with tissue frozen without pre-cooling (77.1% and 60.2 % in Groups 1 and 3, respectively, P_{1, 3-2, 4} <0.05).

Conclusions

Long time (24 h) cooling of ovarian tissue to 5°C before cryopreservation decreased translocation of phosphatidylserine that evidences about increases the viability of the cells in the tissue after thawing.     

Cryoprotectant-free cryopreservation of human spermatozoa by vitrification and freezing in vapor: effect on motility, DNA integrity, and fertilization ability

Human spermatozoa can be successfully cryopreserved avoiding the use of cryoprotectants through vitrification at very high cooling rates (up to 7.2 x 10(5) degrees C/min). This is achieved by directly plunging a copper cryoloop loaded with a sperm suspension into liquid nitrogen. After storage, vitrified spermatozoa are instantly thawed by melting in an agitated, warm medium. The goal of the present study was to compare the quality of spermatozoa cryopreserved using this rapid vitrification method with that of spermatozoa cooled relatively slowly by preexposure of the loaded cryoloop to liquid nitrogen vapor (-160 degrees C) with speed in the range 150-250 degrees C/min) before immersion into liquid nitrogen. Both cooling modes led to comparable results in terms of the motility, fertilization ability, and DNA integrity of the warmed spermatozoa. In both cases, instant thawing by melting in a warm medium was essential for successful cryopreservation. Our findings suggest that optimal regimes for the cryoprotectant-free cryopreservation of spermatozoa need not be restricted to very fast cooling before storage in liquid nitrogen, a wide range of cooling rates being acceptable. Herein, we discuss the implications of this finding in the light of the physics of extra- and intracellular vitrification.     

New technology for vitrification and field (microscope-free) warming and transfer of small ruminant embryos

This study was designed to test the efficiency of recently developed vitrification technology followed by microscope-free thawing and transfer of sheep embryos. In a first set of experiments, in vivo derived embryos at the morula to blastocyst stage were frozen in an automated freezer in ethylene glycol, and after thawing and removal of cryoprotectants, were transferred to recipient ewes according to a standard protocol (control group). A second group of embryos were loaded into open-pulled straws (OPS) and plunged into liquid nitrogen after exposure at room temperature to the media: 10% glycerol (G) for 5 min, 10% G+20% ethylene glycol (EG) for 5 min, 25% G+25% EG for 30s; or 10% EG+10% DMSO for 3 min, 20% EG+20% DMSO+0.3M trehalose for 30s. The OPS were thawed by plunging into tubes containing 0.5M trehalose. After this rapid thawing, the embryos were directly transferred using OPS as the catheter for the transplantation process. In a second set of experiments, in vivo derived and in vitro produced expanded blastocysts were vitrified in OPS and then transferred as described above. The lambing rates recorded (59% for the conventionally cryopreserved in vivo derived embryos, 56% for the vitrified in vivo derived embryos, and 20% for the vitrified in vitro produced embryos), suggest the suitability of the vitrification technique for the transfer of embryos obtained both in vivo and in vitro. This simple technology gives rise to a high embryo survival rate and will no doubt have applications in rearing sheep or other small ruminants.     

Potential importance of vitrification in reproductive medicine

As early as 1985, ice-free cryopreservation of mouse embryos at -196 degrees C by vitrification was reported in an attempted alternative approach to cryostorage. Since then, vitrification techniques have entered more and more the mainstream of animal reproduction as an alternative cryopreservation method to traditional slow-cooling/rapid-thaw protocols. In addition, the last few years have seen a significant resurgence of interest in the potential benefits of vitrification protocols and techniques in human-assisted reproductive technologies. The radical strategy of vitrification results in the total elimination of ice crystal formation, both within the cells being vitrified (intracellular) and in the surrounding solution (extracellular). The protocols for vitrification are very simple. They allow cells and tissue to be placed directly into the cryoprotectant and then plunged directly into liquid nitrogen. To date, however, vitrification as a cryopreservation method has had very little practical impact on human-assisted reproduction, and human preimplantation embryo vitrification is still considered to be largely experimental. Besides the inconsistent survival rates that have been reported, another problem is the wide variety of different carriers and vessels that have been used for vitrification. Second, many different vitrification solutions have been formulated, which has not helped to focus efforts on perfecting a single approach. On the other hand, the reports of successfully completed pregnancies following vitrification at all preimplantation stages is encouraging for further research and clinical implementation. Clearly, however, attention needs to be paid to the inconsistent survival rates following vitrification.     

Lake Ladoga Region: human impacts and recent environmental changes

This paper presents a landscape-ecological analysis of Ladoga Region, a 30 000 km² territory around Lake Ladoga, which is the largest lake in Europe. The Region includes the contact zone between the crystalline bedrock of Baltic Shield and the sedimentary rocks of the East European plain, and the transition from Boreonemoral to Southern Boreal (Taiga) forest vegetation; it is characterized by abundance of lakes. The Region is divided into 17 discrete landscape areas that are classified in 6 groups.The author uses the landscape-dynamcal approach to the analysis of recent (from 2–3 to 40–50 yr) environmental changes in the region, caused by human activities: forest felling, fires, peat excavation, agriculture, recreational development etc. The following processes are observed: vegetation successions after clearcutting and selective logging; effects of forest fires on the spreading and conservation of pine forests; consequences of peatland drainage; landscape effects of collective farming and gardening; forest succession from pine and deciduous tree dominance towards spruce dominance (observable in all landscape regions); natural vegetation succession on former Finnish arable lands (two types of scenarios: forest succession or development of sedge meadows).     

Improvement of attachment rate of bovine embryos after transfer with trophoblastic fragments

Fragments of trophoblastic vesicles have been shown to improve embryo development when used in co-culture. We wished to investigate the effect of trophoblastic fragments on establishment of pregnancy after embryo transfer. Embryos (n=129) frozen by direct immersion in liquid nitrogen (Group 1), 82 embryos frozen by the slow freezing method (Group 2) and 55 fresh embryos stored at 20-22 degrees C for 21-22 h (Group 3) were transferred into recipient heifers. The embryos of each group were divided in 2 batches. Batch 1 embryos (control) were transferred without trophoblastic fragments, Batch 2 embryos of each group (experimental) were transferred in combination with frozen-thawed trophoblastic fragments at a rate of 5 to 7 fragments per embryo. The fragments were produced from 16-17 day bovine embryos frozen by vitrification. Two months following the embryo transfers it was established that the attachament rate for Batch 1 embryos in Groups 1, 2, 3 was 33%, 39% and 48%, respectively; for Batch 2 embryos in the same Groups the rate was 55%, 56% and 74%, respectively. We conclude that trophoblastic fragments promote embryo attachment, but their effect does not continue beyond the time of attachment.     

Vitrification of human laser treated blastocysts within cut standard straws (CSS): novel aseptic packaging and reduced concentrations of cryoprotectants

Vitrification of laser treated human blastocysts using reduced concentrations of permeable cryoprotectants was carried out by submerging cut standard straws (CSS) into liquid nitrogen. The CSS were made by cutting a standard 0.25 ml straw at an angle of approximately 45 degrees . After laser assisted hatching, 6 day blastocysts (n=250) were loaded into droplets of approximately 0.75 microl in the CSS and were either plunged directly into liquid nitrogen or first encased in a standard 0.5 ml straw (aseptic technique) before being vitrified. Permeable cryoprotectants (ethylene glycol+Me(2)SO) at concentrations of 15% and 20% v:v were tested for their effect on post warming re-expansion and post transfer pregnancy rates. Our results indicate that the use of reduced concentrations of cryoprotectants and aseptic packaging of blastocysts did not have any statistically significant impact on the study outcomes.     

Cryopreservation of human ovarian tissue: comparison of rapid and conventional freezing

Cryopreservation, which is the most important procedure in ovarian tissue banking, can be divided into two methods: conventional freezing and rapid freezing. In previous study, the higher effectiveness of rapid freezing in comparison with the conventional freezing for human oocytes and embryos was shown. Data on comparison of these two methods for human ovarian tissue are limited. The aim of this study was to compare conventional freezing and rapid freezing for human ovarian tissue. Ovarian tissue fragments from 14 patients were transported to the laboratory within 22–25 h in a special, isolated transport box, which can maintain a stable temperature of between 5 and 8 °C for 36 h. Small pieces of ovarian tissue (1 × 1–1.5 × 0.7–1 mm) were randomly distributed into four groups: Group 1: control, fresh pieces immediately after receiving transport box, Groups 2 and 3: experimental pieces after rapid freezing/warming, and Group 4: experimental pieces after conventional freezing/thawing. All pieces were cultured in vitro for 14 days. The viability of the tissue by in vitro production of hormones and development of follicles after culture was evaluated. The level of estradiol 17-β and progesterone was measured using heterogeneous competitive magnetic separation immunoassay. For histological analysis, the number of viable and damaged follicles was counted. After culture of fresh tissue pieces (Group 1), rapidly frozen/warmed pieces (Groups 2 and 3), and conventionally frozen/thawed pieces (Group 4), the supernatants showed estradiol 17-β concentrations of 358, 275, 331, and 345 pg/ml, respectively, and progesterone concentrations of 3.02, 1.77, 1.99, and 2.01 ng/ml, respectively. It was detected that 96%, 36%, 39%, and 84% follicles for Groups 1, 2, 3, and 4, respectively, were normal. For cryopreservation of human ovarian tissue, conventional freezing is more promising than rapid freezing.     

Effect of temperature on synthesis of polyphosphates in Yersinia pseudotuberculosis and Listeria monocytogenes under starvation conditions

It was found that at low temperature (6-8 degrees C) in the absence of nitrogen supply and at the presence of phosphate ions in the medium, Yersinia pseudotuberculosis and Listeria monocytogenes are able to actively synthesize reserve substances as polyphosphates. Most of the bacterial polyphosphates are alkali-soluble, especially at the preliminary stage of cell growth (lag-phase). This is proved by electron microscopic studies of ultrastructure of model microorganisms. During a long starvation period under conditions of carbon and energy source deficit, L. monocytogenes and Y. pseudotuberculosis consume this biopolymer for biosynthetic and bioenergetic processes.