Obtaining an Interspecific Hybrid of Cranes by Artificial Insemination with Frozen–thawed Semen

Studies of artificial insemination of cranes and cryoconservation of their semen have been carried out in the nursery of rare species at the Oka Biosphere Reserve for many years. The criterion of successful cryoconservation of the semen is the obtaining of fertilized eggs after artificial insemination by the thawed semen. An experiment is described on artificial insemination of females of the white-naped crane Grus vipio by the frozen–thawed semen of the Siberian white crane G. leucogeranus after one-year storage of semen in liquid nitrogen. As a result, an interspecific hybrid of cranes was obtained, which confirmed the possibility of producing a bank of cryoconserved crane semen. The use of the white-naped crane females was due to the absence of conspecific males and unavailability of Siberian white crane females. Problems of artificial insemination and cryoconservation of semen of rare crane species are discussed.     

Obtaining an Interspecific Hybrid of Cranes by Artificial Insemination with Frozen–thawed Semen

Studies of artificial insemination of cranes and cryoconservation of their semen have been carried out in the nursery of rare species at the Oka Biosphere Reserve for many years. The criterion of successful cryoconservation of the semen is the obtaining of fertilized eggs after artificial insemination by the thawed semen. An experiment is described on artificial insemination of females of the white-naped crane Grus vipio by the frozen–thawed semen of the Siberian white crane G. leucogeranusafter one-year storage of semen in liquid nitrogen. As a result, an interspecific hybrid of cranes was obtained, which confirmed the possibility of producing a bank of cryoconserved crane semen. The use of the white-naped crane females was due to the absence of conspecific males and unavailability of Siberian white crane females. Problems of artificial insemination and cryoconservation of semen of rare crane species are discussed.     

Factors affecting the viability and properties of microorganisms in different preservation methods

The review of literature devoted to the influence of the different methods of longterm preservation on the survival, physiological and biochemical properties of microorganisms: at low and ultralow temperatures, freeze-drying, drying, storage under the mineral oil etc. is given. The microorganisms viability depends on their nature, age and density of population, storage and recovery conditions of cells. Some features of the industrial microorganisms storage have been marked. The different hypotheses concerning the mechanism of preservation, injury and reactivation of microorganisms under the action of external factors during their storage are being discussed.     

Cryopreservation of the unicellular marine alga, Nannochloropsis oculata

In microalgal culture collections, as in many biological resource centres, cryoconservation is the most attractive method for the long-term, secure storage of living material. Nannochloropsis oculata, a marine unicellular alga, is of interest in the field of biotechnology due to its high lipid content. Of various cryoprotectants tested for their toxicity and for their ability to prevent cryoinjury, glycerol (final concentration 1.1 M) was the most efficient. When glycerol-treated cultures were submitted to a strictly regulated cooling rate (-3 degrees C min(-1)), they attained the control culture density within 13 d after thawing.     

Phosphatidylcholine and phosphatidylethanolamine derivatives, membrane fluidity and changes in the lipolytic activity of ram spermatozoa plasma membranes during cryoconservation

1. A decrease of the alkenyl-acyl derivatives and an increase of the diacyl derivatives of PC and PE were observed after cryoconservation. 2. A diminution of membrane-bound phospholipase A2 activity was observed after cryoconservation. The activity of neutral sphingomyelinase remained unchanged. 3. The enrichment of plasma membranes with DPPC as well as the addition of the cryoprotector of Nagase-Niwa were observed to protect the membranes from fluidization.     

工业模式微生物膜有序性的活细胞定量分析

作为一种相位敏感的荧光探针,Di-4-ANEPPDHQ可以特异性标记膜的有序相和无序相,在理论上可以对细胞膜的有序性进行定量成像。通过将Di-4-ANEPPDHQ和激光扫描共聚焦显微术相结合,对多种具有代表性的工业模式微生物进行了有序相和无序相活细胞成像,结合极性归一化数值的统计比较,最终实现对上述工业模式微生物细胞膜有序性的定量分析,为细胞膜工程提供了一种直观且快速的活细胞检测方法。     

工业微生物酸胁迫的耐受机制及改造途径

工业微生物在发酵生产过程中会面对发酵环境和自身产生的各类酸性物质,而这些酸性物质会影响工业微生物的生长和代谢,即产生酸胁迫。微生物通过调控胞内质子浓度、保护和修复生物大分子、改变细胞膜组分以及整体水平调控等耐受机制来应对酸胁迫。结合酸胁迫的各种耐受机制,利用自然筛选和人工改造的方法提高工业微生物的抗酸胁迫能力,为构建出更能适应工业生产条件的菌株提供理论依据。     

Study of DNA integrity in somatic cells and spermatozoa by single-cell gel electrophoresis with silver-nitrate staining

In the present work, genome instability in human and bovine peripheral blood lymphocytes and spermatozoa was studied by the method of DNA microelectrophoresis with subsequent staining of single cells with silver nitrate. A comparative analysis of the types of damage to human and bovine lymphocytes and spermatozoa genomes was performed. In the group of healthy donors, the spontaneous frequency of DNA damage revealed by single cell DNA microelectrophoresis did not exceed 9% and amounted, on average, to 4.8 ± 1.2%. In studying the effect of the duration of cryoconservation on bovine spermatozoa, no significant changes were revealed between the group of bulls whose spermatozoa were stored for less than one year (3.1 ± 0.9%) and the group of animals whose spermatozoa were under conditions of cryoconservation for more than 20 years (4.3 ± 0.5%). From the obtained single-cell DNA microelectropheretic data on the types and frequencies of DNA damage, a conclusion was made regarding the possibility of using a light variant based on cell staining with silver nitrate for the detection of genome instability, not only in somatic, but also in reproductive, cells.     

生产聚羟基脂肪酸酯的微生物细胞工厂

聚羟基脂肪酸酯(PHA)是一类由微生物合成的、生物可再生、生物可降解、具有多种材料学性能的高分子聚合物,在很多领域有着广泛的应用前景。以下从辅酶工程、代谢工程、微氧生产等方面综述了微生物法生产PHA的研究进展,并对利用PHA合成基因提高基因工程菌的代谢潜能进行了讨论。     

Preservation of parenchymal and stromal progenitors in a cryopreserved human fetal liver

The presence of a viable population of hepatoblasts, epithelial blast cells, and endothelial and mesenchymal cells in a cryoconserved suspension of human fetal liver (FL) cells was shown. Epithelial-mesenchymal hepatoblast transformation in a cell culture was found. A possibility to apply the previously developed method of cryoconservation of human FL hematopoietic cells in the first trimester of gestation for the preservation of the heterogenic population of parenchymal and stromal cells was shown.     

Short Term Preservation of Hide Using Vacuum: Influence on Properties of Hide and of Processed Leather

The objective of this work was to investigate vacuum influence on hide preservation time and how it affects hide structure. It was established that vacuum prolongs the storage time without hide tissue putrefaction up to 21 days when the storage temperature is 4°C. The microorganisms act for all storage times, but the action is weak and has no observable influence on the quality of hide during the time period mentioned. The hide shrinkage temperature decrease is negligible, which shows that breaking of intermolecular bonds does not occur. Optical microscopy, infrared spectroscopy and differential scanning calorimetry also did not show any structural changes which can influence the quality of leather produced from such hide. The qualitative indexes of wet blue processed under laboratory conditions and of leather produced during industrial trials are presented. Indexes such as chromium compounds exhaustion, content of chromium in leather, content of soluble matter in dichloromethane, strength properties, and shrinkage temperature were determined. Properties of the leather produced from vacuumed hide under industrial conditions conformed to the requirements of shoe upper leather.     

Biomachining: Preservation of Acidithiobacillus ferrooxidans and treatment of the liquid residue

Biomachining has become a promising alternative to micromachining metal pieces, as it is considered more environmentally friendly than their physical and chemical machining counterparts. In this research work, two strategies that contribute to the development of this innovative technology and could promote its industrial implementation were investigated: preservation of biomachining microorganisms (Acidithiobacillus ferrooxidans) for their further use, and making valuable use of the liquid residue obtained following the biomachining process. Regarding the preservation method, freeze‐drying, freezing, and drying were tested to preserve biomachining bacteria, and the effect of different cryoprotectants, storage times, and temperatures was studied. Freezing at –80°C in Eppendorf cryovials using betaine as a cryoprotective agent reported the highest bacteria survival rate (40% of cell recovery) among the studied processes. The treatment of the liquid residue in two successive stages led to the precipitation of most of the total dissolved iron and divalent copper (99.9%). The by‐products obtained (iron and copper hydroxide) could be reused in several industrial applications, thereby enhancing the environmentally friendly nature of the biomachining process.     

Aspects réglementaires et organisationnels de l’activité d’autoconservation. Le point de vue du juriste

At the present time, legal texts in application of bioethics laws only briefly mention cryopreservation. The conditions of cryopreservation differ according to the type of tissue stored: while cryopreservation of oocytes and ovarian tissue corresponds to the field of biomedical research, semen cryopreservation corresponds to medically assisted procreation. Cryopreservation activity is more clearly defined in the draft revision of the bioethics law. Concerning the cryoconservation of ovarian tissue and oocytes, these difficulties result in particular in this activity’s mixed nature. It is located indeed, halfway between research and care. These two spheres of medical activity are subjected to their own distinct and exclusive, and their application is conditioned by the qualification of the implemented act. However, this qualification is dubious here, because of, in particular, the impossibility of determining which acts of sampling, cryoconservation and use might be concerned with a research protocol. Concerning the cryoconservation of sperm, the texts, first of all, seem to assimilate the activity of cryoconservation within an activity of assisted medical procreation. However, such assimilation would be equivalent to the impossibility of its implementation, because of the difficulty of respecting all of the legal conditions of assisted medical procreation. However, another more favourable interpretation of the provisions seems to be possible. Taking into consideration these uncertainties, contradictions and difficulties, the legislator intervened and devoted, by the widening of the indications of assisted medical procreation, the activity of cryoconservation in the project of revision of the laws of bioethics. He however did not solve all the difficulties. These cryoconserved elements can be restored only to the depositor. Indeed, the texts make obstacle to a delivery for a third party of the cryoconserved sperm, whatever the moment. They can also be used. This use can consist of assisted medical procreation, which is the first finality of cryoconservation — but it could only be implemented in respect of the whole of the legal conditions. It seems that the cryoconserved elements could also be used within the framework of research, whatever its nature (biomedical or not) and the moment of its implementation (while the person is alive or after his death). Its implementation should however be subjected to prior agreement and expressed while the person, whose elements were preserved, is alive. Such a use is expressly made possible in the project of revision of the laws of bioethics. Following a partial use or a lack of such a use, the destruction of the cryoconserved elements can be considered, as well as the continuation of the cryoconservation. However, these hypotheses raise difficulties that have not yet been resolved in the draft revision of the bioethics law.     

Effect of Cryoconservation on Seed Germination of Rare Tropical Orchids

In search of a procedure for cryoconservation, seeds of five species of tropical orchids and of one intergeneric hybrid were germinatedin vitroas depended on illumination. The other portion of seeds was frozen in liquid nitrogen (–196°C) and germinated after thawing. The viability of seeds, the percentage and rate of germination, as well as the growth rate of the developed protocorms were evaluated. At early stages of development, light inhibited growth in most species examined. The storage of seeds in liquid nitrogen had no negative effect on growth and development of protocorms and juvenile plants. The feasibility of the conservation of mature seeds of some tropical orchids by freezing in liquid nitrogen was demonstrated, which offers opportunities for creating a cryobank of orchids.     

Recording of Processes of Hemoglobin Oxidation and Cryoprotective Effect of Glycerol

It was shown earlier that functional properties of hemoglobin changed during storage. Our goal was to evaluate processes of hemoglobin autooxidation and to develop simple methods for conservation of hemoglobin samples. It is revealed that cryoprotective effect of glycerol rapidly rises with increase of its concentration up to 5% for hemoglobins of all 4 studied species of sturgeon fish, sterlet, Russian sturgeon, starred sturgeon, and great sturgeon. The cryoprotective effect of glycerol is the highest on hemoglobin of the starred sturgeon, the lowest, of the sterlet. The results of this study allow proposing the method for cryoconservation of the sturgeon fish hemoglobin with addition of glycerol (up to 5% ) to the frozen solution of hemoglobin.     

Studies of free radical-mediated cryoinjury in the unicellular green alga Euglena gracilis using a non-destructive hydroxyl radical assay: a novel approach for developing protistan cryopreservation strategies

The development of cryoconservation methods for the long-term storage of algal cultures is important for the ex situ preservation of biological diversity and the maintenance of genetic stability within this group of important organisms. However, as many unicellular algae are recalcitrant to cryogenic storage, this study aims to evaluate the role of oxidative stress in cryoinjury. A non-invasive, non-destructive assay method previously applied to animal cells has been developed to evaluate free radical mediated oxidative stress in Euglena gracilis exposed to different cryopreservation treatments. The procedure employs dimethyl sulphoxide as a probe for the hydroxyl radical. Adopting this approach it was possible to identify those components of the cryopreservation protocol which were the most damaging. These were identified as preparative centrifugation and sub-zero freezing treatments. Poststorage survival in E. gracilis was significantly (P < 0.05) enhanced when the chelating agent desferrioxamine was included in the recovery medium whilst methane production was significantly (P < 0.004) reduced, suggesting that the additive was capable of ameliorating oxidative stress. The potential of using novel, exogenous antioxidant treatments developed for medical applications and applying them to enhance cryopreservation tolerance in recalcitrant unicellular algae is discussed.     

An immobilized hydrogenase from Alcaligenes eutrophus H-16

Alcaligenes Eutrophus H-16 was grown in continuous culture under conditions which induced hydrogenase production. The hydrogenase enzyme was extracted, partially purified and immobilized on porous glass. This enzyme was then studied both in solution and in immobilized form as a possible candidate for a number of industrial applications. It proved to have a stability (storage and operational) which was highly temperature dependent. Temperatures near freezing caused the enzyme to retain its activity for long periods of time. Although its kinetics were more favorable at elevated temperatures of up to 40 degrees C, the loss of stability outweighed this gain substantially. The effects of buffer type and pH on enzyme activity were also studied. This enzyme has only a modest sensitivity to destruction by oxygen during storage, in contrast to hydrogenases produced by several other microorganisms.     

微生物耐受乙醇与丁醇机制及其在生物燃料生产与生物转化中的应用

生物法获取乙醇与丁醇过程中有机溶剂的毒性是生产菌重要环境胁迫因素之一,且当有机溶剂超过一定浓度时便会抑制微生物的生长,甚至引起微生物的死亡,因此提高工业微生物的有机溶剂耐受性对工业生产具有重要的意义。对微生物乙醇及丁醇耐受机制的研究可为选育具有较强溶剂耐受菌提供理论基础。本文系统介绍了微生物耐受乙醇与丁醇的机制,并对其在生物燃料生产及生物转化中面临的机遇与挑战等问题进行简要的评述。     

Production of citric acid by immobilized dried reactivatedAspergillus niger

Summary Immobilization followed by drying was fried as a new technique for obtaining small beads which might be more suitable for industrial fermentations and for the storage of the immobilized microorganisms.Aspergillus niger NRRL 2270 was used to produce citric acid in free, immobilized, and immobilized dried reactivated (IDR) forms. The productivity based on the bead volume used increased several folds with the use of IDR cells although the absolute level of citric acid did not increase.