Search

India is a treasure trove of biological diversity with its plant genetic resources playing a crucial role in the crop improvement serving as the foundation for the country's sustainable food and nutritional security. India's in vitro genebank (IVG) is part of the National Genebank at the Indian Council of Agricultural Research-National Bureau of Plant Genetic Resources (ICAR-NBPGR). The IVG houses distinctive multi-crop repository that utilizes several tissue culture techniques for short- to medium-term storage in in vitro active genebank (IVAG) and cryoconservation approaches for long-term storage in in vitro base genebank (IVBG). In IVAG, germplasm is conserved under normal and slow growth conditions with a subculture period of 1–36 months depending on the species/genotype and conservation approach. Currently, the IVAG holds 2,038 germplasm accessions (69 genera and 171 species) from six crop groups, viz. (a) tropical fruit crops (449), (b) temperate and minor tropical fruit crops (408), (c) tuber crops (530), (d) bulbous and ornamental crops (187), (e) medicinal and aromatic plants (232), and (f) spices and industrial crops (232). For long-term conservation, in vitro produced explants of various species are cryopreserved at the IVBG in liquid nitrogen. Utilizing various cryoconservation procedures, 347 accessions from several crop groups have been successfully conserved in the IVBG. Over the past four decades, in vitro conservation has been accomplished by the above mentioned cutting-edge techniques. This report highlights the efforts and achievements of the National Genebank in conserving horticultural genetic resources through in vitro and cryoconservation. [ABSTRACT FROM AUTHOR]

Copyright of Basic & Clinical Andrology is the property of BioMed Central and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

This study investigated the thermal properties of potato and hop pollen for cryopreservation and subsequent cross-breeding. Phase transitions and frozen water content in selected pollen samples were measured using a differential scanning calorimeter (DSC). Unlike hop pollen, potato pollen showed high variability in thermal properties and water content. Three specific types of pollen samples based on their thermal characteristics and water content were distinguished by DSC in potato: (1) 'glassy', with a water content lower than 0.21 g water per g dry matter; (2) 'transient', with a water content between 0.27 and 0.34 g of water per g of dry matter; (3) 'frozen', with a water content higher than 0.34 g of water per g of dry matter. Only the 'glassy' pollen samples with a low water content showed suitable properties for its long-term storage using cryopreservation in potato and hops. Cryopreservation of pollen did not significantly reduce its viability, and cryopreserved pollen was successfully used to produce both potato and hop hybrids. The results indicate that cryopreservation is a feasible technique for the preservation and utilization of pollen of these crops in the breeding process. [ABSTRACT FROM AUTHOR]

• Biopreservation is reviewed from a unique perspective according to the order of temperature. • The causes of cryo-injury of different biological materials are listed in detail. • Detailed preservation and damage mechanisms of different biomaterials are stated. Low-temperature biopreservation is widely used for the storage of medications. As biochemical reactions in cells are slow at low temperatures, they can be stored for longer periods. However, cryopreservation can result in cryo-injury. Therefore, determining how to achieve long-term biopreservation without causing injury has become an essential research topic. Biological tissues should be stored at different temperature zones depending on the purpose of storage and cell characteristics. In order to achieve better storage efficiency, it is critical to investigate and review the best storage temperature for various biomaterials. The mechanisms of cryo-injury and biopreservation are discussed in this review, and the reasons for the preservation of biological tissues at different temperature zones are also reviewed. Based on the review, the storage characteristics of different temperature ranges are summarized, and the future development of biological storage is prospected. This review has some reference significance for biostorage technology. [ABSTRACT FROM AUTHOR]

As part of a comprehensive program for registering and preserving the gene pool of Russian varieties in the VIR genebank, initiated at the N.I. Vavilov All-Russian Institute of Plant Genetic Resources, nomenclatural standards were assigned to potato cultivars ‘Alâska’, ‘Argo’, ‘Bravo’, ‘Irbitskij’, ‘Legenda’, ‘Lûks’, ‘Terra’, and ‘Šah’, and a voucher specimen was prepared for pre-cultivar ‘Bagira’. All of them were bred at the Ural Federal Agrarian Research Centre (Ural Branch of the Russian Academy of Sciences). Nomenclatural standards of these cultivars are preserved in the Nomenclatural standard collection at the Herbarium of cultivated plants and their wild relatives and weeds (VIR Herbarium, WIR). For each of them, a genetic passport was developed using DNA preparations isolated from plant material donated by the cultivar author to the VIR herbarium. The genetic passport, which includes information on the allelic composition of eight chromosome-specific microsatellite loci, is supplemented by the molecular screening data with markers of 11 R-genes for resistance to pests, as well as the data on the types of cytoplasm. The author of cultivars also supplied VIR with samples of virus-free in vitro plants. Microplants of eight Ural cultivars and one pre-cultivar were genotyped using the same SSR markers and included in the VIR in vitro collection, and then in the cryopreservation program. Most of the accessions were characterized by a relatively high, above 39%, level of post-cryogenic regeneration. Frozen explants of the genotyped Ural cultivars (‘Alâska’, ‘Argo’, ‘Legenda’, ‘Terra’, and ‘Šah’) and pre-cultivar ‘Bagira’ were placed for long-term storage in the VIR Cryobank.

Background. The article presents the results of the studies carried out within the framework of a complex program for the long-term conservation of genotyped accessions of advanced potato cultivars in the cryobank of the N.I. Vavilov All-Russian Institute of Plant Genetic Resources (VIR). These accessions are genetically identical to the nomenclature standards of the corresponding cultivars.Materials and methods. The research material included 24 accessions from the in vitro collection of VIR, corresponding to the nomenclature standards of Russian potato cultivars developed in different breeding centers of the Russian Federation. Cryopreservation was carried out using the droplet vitrification method, several stages of which were modified in the Department of Biotechnology of VIR.Results and discussion. The frequency of post-cryogenic regeneration for the analyzed 24 cultivars in control variants with short-term immersion of explants in liquid nitrogen varied from 20 to 47.3%, and in 9 accessions it exceeded 39% – the current level of reliable long-term preservation of samples in cryobanks. The data of the statistical analysis failed to reveal a significant effect of the genotype or the calendar dates of different experiment replications on the regeneration ability. These 24 accessions were added to the potato cryocollection stored in the VIR cryobank. An analysis of the composition and structure of VIR’s potato cryocollection, which currently includes 140 accessions, is presented.

La conservation à l’état liquide ainsi que la cryoconservation de la semence de bélier sont des étapes clés dans la réussite de l'insémination artificielle. Cependant, la manipulation de la semence entraîne généralement une diminution progressive de sa qualité et de sa capacité de fécondation. Dans les deux méthodes de conservation, les chocs thermique et osmotique ainsi que le choix inapproprié du dilueur pourraient conduire aux dommages oxydatifs induits par les espèces réactives de l'oxygène sur les spermatozoïdes. Par conséquent, des études antérieures se sont concentrées sur la supplémentation de dilueurs par des antioxydants exogènes afin de réduire le stress oxydatif produit lors de la conservation du sperme de bélier. Récemment, des biomolécules provenant de produits naturels ont été additionnés au dilueurs. Cet article vise à présenter en détail les facteurs influençant la qualité de la semence de bélier lors de la conservation en sperme frais ou congelé. En outre, les résultats des études portant sur l'utilisation des extraits ou des biomolécules naturelles à différentes doses sont discutés. Précisément, une mise à jour concernant leurs effets sur la qualité du sperme et sa capacité de fécondation in vivo ou in vitro est fournie dans cette revue.

This study investigated the thermal properties of potato and hop pollen for cryopreservation and subsequent cross-breeding. Phase transitions and frozen water content in selected pollen samples were measured using a differential scanning calorimeter (DSC). Unlike hop pollen, potato pollen showed high variability in thermal properties and water content. Three specific types of pollen samples based on their thermal characteristics and water content were distinguished by DSC in potato: (1) ‘glassy’, with a water content lower than 0.21 g water per g dry matter; (2) ‘transient’, with a water content between 0.27 and 0.34 g of water per g of dry matter; (3) ‘frozen’, with a water content higher than 0.34 g of water per g of dry matter. Only the ‘glassy’ pollen samples with a low water content showed suitable properties for its long-term storage using cryopreservation in potato and hops. Cryopreservation of pollen did not significantly reduce its viability, and cryopreserved pollen was successfully used to produce both potato and hop hybrids. The results indicate that cryopreservation is a feasible technique for the preservation and utilization of pollen of these crops in the breeding process.

Key message: The developed protocol for organogenesis, in vitro plantlet production, and cryopreservation opens the possibility for mass propagation of hybrid pine (Pinus elliottii var. elliottii x Pinus caribaea var. hondurensis). The low seed production of the interspecific hybrid Pinus elliottii var. elliottii x Pinus caribaea var. hondurensis restricts its commercial expansion, making it necessary to ensure efficient cryopreservation and a propagation protocol with no genetic variability. Mature zygotic embryos (MZEs) were cultured in Murashige and Skoog (MS) medium containing 6-benzyladenine (BA) and thidiazuron (TDZ). After 45 days in culture, the highest rate of regeneration (86.7 ± 8.8%) and the maximum number of differentiated buds per responsive explant (15.5 ± 2.8) were achieved from explants cultivated on MS with BA and TDZ (0.5 μM each). Cryopreservation of zygotic embryos using a simple desiccation step and a direct immersion into liquid nitrogen did not affect regeneration and would enhance embryo storage duration. Half-strength MS enriched with sucrose (0.09 M) and gelled with gellan gum (4 g L− 1) under forced ventilation culture was used for shoot elongation. Subsequently, 73 ± 6.7% of shoots produced roots after pretreatment with 1.25 mM indole-3-butyric acid solution for 5 min and culture on quarter-strength MS with sucrose (0.045 M). The regenerated plantlets were successfully transferred to ex vitro conditions. The procedure took 160 days and comprised the adventitious bud formation from cryopreserved MZEs, shoot elongation, rooting, and plantlet acclimation. Considering that water deficit is the major strain during forest establishment, a controlled experiment was carried out to determine the competence of plantlets to overcome this stress. Next, field studies assessed the survival rate and growth of 16-month-old plants. Our results indicated that the field performance of tissue-culture-derived plants is similar to seedlings and rooted cutting plants. Additionally, inter-simple sequence repeat marker analysis revealed the genetic uniformity among the in vitro-raised plants, demonstrating the reliability and validity of the procedure. Thus, the developed regeneration and cryopreservation protocol for mature zygotic embryo explants is a valuable alternative for breeding programs and commercial P. elliottii x P. caribaea propagation. [ABSTRACT FROM AUTHOR]

Copyright of Kosmos is the property of Polish Copernicus Society of Naturalists and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Somatic embryogenesis, as a promising biotechnological tool for many conifer trees, has never been applied for the Abies nebrodensis species. Although all the encouraging results previously obtained by the EU LIFE (European LIFE program) funded projects in over ten years, the critically endangered Sicilian fir remains alarmingly close to extinction. In this study, we reported the first protocol of somatic embryogenesis obtained from mature zygotic embryos of the Abies nebrodensis. Seeds from Abies adult trees with specific identification numbers (IN) were collected and full seeds were identified by X-ray. Different experiments were carried out for callus initiation, from both zygotic immature and mature embryos, testing different culture media. The immature embryos did not give embryogenic tissue (ET). Embryogenic callus (EC) was successfully induced from mature embryos with variable frequencies (0–40%). Schenk and Hilderbrandt (SH) was the most suitable initiation medium where the obtained callus initiation rate reached up to 40% for IN7 (first experiment). 6-benzylaminopurine (BAP) showed to be essential to induce EC (second experiment). IN8 presented the highest callus initiation rate (40%) among all tested donor trees, whereas IN13 recorded the lowest rate with 4% (third experiment). ET maturation from each singular embryo of IN7, IN8, IN10 and IN21 was successfully achieved in SH medium containing 37,83 µM abscisic acid (ABA), 8% of polyethylene glycol (PEG-4000) and 4% maltose. The encapsulation technology was assessed on the obtained ET and its proliferation was observed after encapsulation. Key message: A protocol for somatic embryogenesis from mature embryos of the Abies nebrodensis was achieved. [ABSTRACT FROM AUTHOR]

Introduction: This study describes a variation of the cryodehydration technique, whose first phase follows the same procedures previously described in the literature (fixation, freezing, and sectioning according to established goals (in the longitudinal, horizontal, and transversal planes) as preparation for repeated freeze-thaw cycles. Material and Methods: In the second phase, the anatomical specimens were fully immersed in wood glue and placed in a vacuum chamber, with application of negative pressure. Results: Thus, the once air-filled spaces were suffused with glue and the bubbling and movement of gases from tissue fluids facilitated the seepage of glue into the existing spaces. Conclusion: The obtained material has a unique quality, no shrinkage, and high resistance associated with longer durability for use in theoretical and practical classes and also as museum specimens. [ABSTRACT FROM AUTHOR]

The biopreparation’s effect of algal origin on cells semen’s cryoresistance and the reproductive system function of the sire bulls was established. BioR was influenced significantly (fig. 1) the cryoresistence of the semen material of sires bulls. To the end of the administration period (10 days), the group cured with 0,1 ml/ 100kg living mass /day showed an increase of the spermatic mobility with 69,14%, comparing to the preexperimental period. The group cured with 0,4 ml/ 100kg living mass /day showed an increase of 3,88%. The next 50 days from the ceasing of the preparation administration, the seminal cells mobility increased for the first group (0,1 ml/100 kg living mass/day) with 107,14%, and for the second group (0,4 ml/100 kg living mass/day) this indices value maintains on the stage. The DMA concentration in blood for the first group was 3,74 mmol/l, 10 days after the administration we established an decrease of this indices till 3,12 mmol/l (P

The evidence of cryogenics response of the semen proteins, the influence of BioR administration on homeostasis of constituent gametes proteomics and on the cryobiological indexes of bull semen material was studded. The investigation has been performed on bulls from the Black Spotted breed of Moldavian type, maintained during the investigation in adequate conditions from the point of view of microclimate and fodder. The biopreparation administration have been done daily during 10 days in volume of 0,2 ml/100 kg living mass/day. Structural proteins of gametes posed the resistance given the influence of ultra low temperature (-196°C), content of totals proteins in the bull semen material denote no difference between the value of this parameter in the raw and cry preserved-thawed bull gametes. Both, in the raw and thawed semen cells the most rate occupy the hydrophilic proteins, After semen conservation-thawing process, it was observed a tendency of the diminution of hydrophilic proteins (- 3,35%) and an increase of the basophilic proteins (+ 2,78 %). In the raw gametes prevail γ-globulins rate; conservation and thawing process of the semen material was associated by an increase of the albumins rate (+ 34,63%) in semen cells; the rate of other three proteomic fractions: α-, β - and γ-globulins was decreased given theirs value registered in raw gametes. After the intramuscular administration of BioR preparation during 10 days on the sire bulls have been certified any modification of the studded proteomic fractions rate in thawed bull semen cells; albumins rate was decreased with 30,14%, the γglobulins rate was increased with 19,28% in the experimental group; the β- and αglobulins with 8,5% and 2,36%, respectively, given control group. The BioR has an evident influence on the cryobiological specifics features of spermatozoids, such as the seminal cells mobility, the longevity and the survival absolutly index what are intensely influenced.

Long-term conservation of Plant Genetic Resources (PGR) is a key priority for guaranteeing food security and sustainability of agricultural systems for current and future generations. The need for the secure conservation of genetic resources collections ex situ is critical, due to rapid and extreme climatic changes which are threatening and reducing biodiversity in their natural environments. The International Potato Center (CIP) conserves one of the most complete and diverse genetic resources collections of potato, with more than 7500 accessions composed of 4900 cultivated potato and 2600 potato wild relative accessions. The clonal conservation of cultivated potato, principally landraces, through in vitro or field collections is indispensable to maintain fixed allelic states, yet it is costly and labor-intensive. Cryopreservation, the conservation of biological samples in liquid nitrogen (-196°C), is considered the most reliable and cost-efficient long-term ex-situ conservation method for clonal crops. Over the last decade, CIP has built one of the largest potato cryobanks worldwide, cyopreserving more than 4000 cultivated potato accessions which represents 84% of the total cultivated potato collection currently conserved at CIP. In approximately, four years the entire potato collection will be cryopreserved. The development of an applied, robust cryopreservation protocol for potato, serves as a model for other clonally maintained crop collections. The CIP cryobank designs experiments with a high number of genetically diverse genotypes (70-100 accessions, seven cultivated species), to obtain reliable results that can be extrapolated over the collection as genotypes can often respond variably to the same applied conditions. Unlike most published reports on cryopreservation of plants, these large-scale experiments on potato are unique as they examine the acclimatization process of in vitro plants prior to, as well as during cryopreservation on up to ten times the number of genotypes conventionally reported in the published literature. As a result, an operational cryopreservation protocol for potato has advanced that works well across diverse potato accessions, not only with reduced processing time and costs, but also with an increased average full-plant recovery rate from 58% to 73% (+LN) for routine cryopreservation. The present article describes the composition of CIP’s cryobank, the cryopreservation protocol, methodology for the dynamic improvement of the operational protocol, as well as data collected on regeneration from long term cryopreserved potatoes. [ABSTRACT FROM AUTHOR]

Long-term conservation of Plant Genetic Resources (PGR) is a key priority for guaranteeing food security and sustainability of agricultural systems for current and future generations. The need for the secure conservation of genetic resources collections ex situ is critical, due to rapid and extreme climatic changes which are threatening and reducing biodiversity in their natural environments. The International Potato Center (CIP) conserves one of the most complete and diverse genetic resources collections of potato, with more than 7500 accessions composed of 4900 cultivated potato and 2600 potato wild relative accessions. The clonal conservation of cultivated potato, principally landraces, through in vitro or field collections is indispensable to maintain fixed allelic states, yet it is costly and labor-intensive. Cryopreservation, the conservation of biological samples in liquid nitrogen (-196°C), is considered the most reliable and cost-efficient long-term ex-situ conservation method for clonal crops. Over the last decade, CIP has built one of the largest potato cryobanks worldwide, cyopreserving more than 4000 cultivated potato accessions which represents 84% of the total cultivated potato collection currently conserved at CIP. In approximately, four years the entire potato collection will be cryopreserved. The development of an applied, robust cryopreservation protocol for potato, serves as a model for other clonally maintained crop collections. The CIP cryobank designs experiments with a high number of genetically diverse genotypes (70-100 accessions, seven cultivated species), to obtain reliable results that can be extrapolated over the collection as genotypes can often respond variably to the same applied conditions. Unlike most published reports on cryopreservation of plants, these large-scale experiments on potato are unique as they examine the acclimatization process of in vitro plants prior to, as well as during cryopreservation on up to ten times the number of genotypes conventionally reported in the published literature. As a result, an operational cryopreservation protocol for potato has advanced that works well across diverse potato accessions, not only with reduced processing time and costs, but also with an increased average full-plant recovery rate from 58% to 73% (+LN) for routine cryopreservation. The present article describes the composition of CIP’s cryobank, the cryopreservation protocol, methodology for the dynamic improvement of the operational protocol, as well as data collected on regeneration from long term cryopreserved potatoes.

Due to the constant development of medicine and biotechnology, including the in vitro procedure, more and more infertile couples can fulfil their desire to have a child. Traditional rules of family law concerning the determination of status familiae of such children have then turned out to be insufficient. The situation is even more complicated when IVF procedure assumes the use of donated genetic material or the involvement of a surrogate mother. Therefore in the field of family law the intention to "create" a child starts to play quite an important role. Family law is then undergoing far-reaching transformations, especially in those countries where same-sex couples are eligible to start IVF procedures (Great Britain, USA, Germany, Austria). [ABSTRACT FROM AUTHOR]

Due to the constant development of medicine and biotechnology, including the in vitro procedure, more and more infertile couples can fulfil their desire to have a child. Traditional rules of family law concerning the determination of status familiae of such children have then turned out to be insufficient. The situation is even more complicated when IVF procedure assumes the use of donated genetic material or the involvement of a surrogate mother. Therefore in the field of family law the intention to "create" a child starts to play quite an important role. Family law is then undergoing far-reaching transformations, especially in those countries where same-sex couples are eligible to start IVF procedures (Great Britain, USA, Germany, Austria). [ABSTRACT FROM AUTHOR]

Copyright of Basic & Clinical Andrology is the property of BioMed Central and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Modern biobanks maintain valuable living materials for medical diagnostics, reproduction medicine, and conservation purposes. To guarantee high quality during long-term storage and to avoid metabolic activities, cryostorage is often conducted in the N2 vapour phase or in liquid nitrogen (LN) at temperatures below − 150 °C. One potential risk of cryostorage is microbial cross contamination in the LN storage tanks. The current review summarises data on the occurrence of microorganisms that may compromise the safety and quality of biological materials during long-term storage. We assess the potential for the microbial contamination of LN in storage tanks holding different biological materials based on the detection by culture-based and molecular approaches. The samples themselves, the LN, the human microbiome, and the surrounding environment are possible routes of contamination and can cause cross contaminations via the LN phase. In general, the results showed that LN is typically not the source of major contaminations and only a few studies provided evidence for a risk of microbial cross contamination. So far, culture-based and culture-independent techniques detected only low amounts of microbial cells, indicating that cross contamination may occur at a very low frequency. To further minimise the potential risk of microbial cross contaminations, we recommend reducing the formation of ice crystals in cryotanks that can entrap environmental microorganisms and using sealed or second sample packing. A short survey demonstrated the awareness for microbial contaminations of storage containers among different culture collections. Although most participants consider the risk of cross contaminations in LN storage tanks as low, they prevent potential contaminations by using sealed devices and − 150 °C freezers. It is concluded that the overall risk for cross contaminations in biobanks is relatively low when following standard operating procedures (SOPs). We evaluated the potential sources in detail and summarised our results in a risk assessment spreadsheet which can be used for the quality management of biobanks. Key points: • Identification of potential contaminants and their sources in LN storage tanks. • Recommendations to reduce this risk of LN storage tank contamination. • Development of a risk assessment spreadsheet to support quality management. [ABSTRACT FROM AUTHOR]

Copyright of Archives of Razi Institute is the property of Institut Razi and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Cryopreservation provides long-term storage of the gene pool of potato varieties in cryobanks at extremely low temperatures. Currently, droplet vitrification is the most widely used method for cryopreservation of potato varieties, which is constantly improving to increase the regeneration rates of the stored plant material. Different modifications of this method are used in the world’s leading potato genebanks. This paper presents the results of studying the effect of cultivation conditions after plunging into liquid nitrogen and thawing of shoots tips and axillary buds of in vitro plants on their postcryogenic recovery. The droplet-vitrification method modified at VIR was used for cryopreservation. The factor “prolonged dark incubation of explants” did not have a significant effect on the frequency of post-cryogenic regeneration of the studied varieties except for one variety (Krepysh), for which a significant increase in the regeneration rate was observed for the shoot tips cultivated in the darkness compared to the cultivation under the photoperiod 16/8 hours (light/darkness). The frequency of post-cryogenic regeneration of shoot tips was higher than that of the axillary buds for all varieties; however, these differences were significant (p < 0.05) only in two cases: for the variety Udacha (a photoperiod of 16/8 hours) and for the variety Krepysh (the dark incubation). The results of two-factor analysis of variance indicate that there is no effect of interaction of factor 1 (prolonged dark incubation) and factor 2 (explant type) on the ability of varieties to post-cryogenic recovery. Taking into account the obtained results, the further cryopreservation of an extended subset of 9 varieties was carried out using shoot tips, which, after freezing-thawing, were cultivated under the photoperiod of 16/8 hours. The frequency of post-cryogenic regeneration of these varieties varied from 30 to 60 %. A significant effect of genotype on postcryogenic recovery has been established. The ability of varieties to regenerate shoots after freezing and thawing was not related to the values of morphogenic indices of in vitro plants. The age of the meriklons (2–4 years) did not significantly affect either the morphogenic indices or the frequency of post-cryogenic regeneration.

Improvements in ex situ storage of genetic and reproductive materials offer an alternative for endangered livestock breed conservation. This paper presents a dataset for current ex situ collections and in situ population for 179 Spanish livestock breeds of seven species, cattle, sheep, pig, chicken, goat, horse and donkey. Ex situ data was obtained via survey administered to 18 functioning gene banks in Spain and relates to the reproductive genetic materials (semen doses) of 210 livestock breeds distributed across the gene banks. In situ data combines CENSUS information with linear regression techniques and relates to the geographic distribution of 179 Spanish autochthonous livestock breeds (2009-2018), and in situ population projections and extinction probabilities (2019-2060). We use a decision variable defining an “acceptable level of risk” that allows decision makers to specify tolerable levels of in situ breed endangerment when taking ex situ collection and storage decisions.

Amphibians are becoming increasingly reliant on captive breeding programs for continued survival. Assisted reproductive technologies including gamete cryopreservation and IVF can help reduce costs of breeding programs, provide insurance against extinction and assist genetic rescue in wild populations. However, the use of these technologies to produce reproductively mature offspring has only been demonstrated in a few non-model species. We aimed to optimise sperm cryopreservation in the threatened frog Litoria aurea and generate mature offspring from frozen-thawed spermatozoa by IVF. We tested three concentrations (1.4, 2.1 and 2.8 M) of the cryoprotectants dimethylsulfoxide (DMSO) and glycerol with 0.3M sucrose. Using DMSO was more likely to result in recovery of sperm motility, vitality and acrosome integrity than glycerol, regardless of concentration, with forward progressive motility being most sensitive to damage. The lowest concentrations of 1.4 and 2.1M provided the best protection regardless of cryoprotectant type. Spermatozoa cryopreserved in 2.1M DMSO outperformed spermatozoa cryopreserved in equivalent concentrations of glycerol in terms of their ability to fertilise ova, resulting in higher rates of embryos hatching and several individuals reaching sexual maturity. We have demonstrated that sperm cryopreservation and subsequent offspring generation via IVF is a feasible conservation tool for L. aurea and other threatened amphibians. [ABSTRACT FROM AUTHOR]

Ionizing radiation is an important environmental factor affecting the dynamics of biospheric processes in the past and present, as well as limiting the spread of life outside the Earth. The effect of radiation on microorganisms has been studied for decades, but studies of the response of natural microbial ecosystems are still scarce. We have studied the effect of 100 kGy gamma irradiation under low pressure (1 Torr) and low temperature (–50°C) on microbial community of the ancient Antarctic permafrost sedimentary rock. After irradiation, the total number of prokaryotic cells determined by epifluorescence microscopy, as well as the number of metabolically active bacterial and archaeal cells detected by fluorescence in situ hybridization remained at the control level, while the number of cultured heterotrophic bacteria decreased by an order of magnitude. Using the multisubstrate testing method, it has been found that the microbial complex retained a high potential metabolic activity and functional diversity after exposure to a combination of extreme physical factors. The resistance demonstrated by the microbial community significantly exceeded the generally accepted estimates of the prokaryotes' radioresistance and indicated an underestimation of the microorganisms' radioresistance in natural habitats and the important role of mineral heterophase environment and irradiation conditions (pressure, temperature). The study confirmed the potential for long-term cryopreservation of viable terrestrial-like microorganisms in the Martian regolith, as well as the possibility of transferring anabiotic life forms as a part of small bodies in the space environment. [ABSTRACT FROM AUTHOR]

The present study provides an efficient cryopreservation protocol for Tulipa tarda cultured in vitro. Apices were excised from bulblets cultivated on MS medium, supplemented with 60 or 90 g l− 1 sucrose. Half of the bulblets were subjected to a cold treatment at 5 °C for 10 weeks, before exposure of the apices to loading solution (LS) and plant vitrification solution 2 (PVS2). Ten weeks after rewarming and culture on recovery medium, 100% regrowth rates were obtained for cold treated explants cultured on 60 g l− 1 sucrose after 30 and 60 min exposure to PVS2. Cold treatment significantly improved the recovery rates of most of the cryopreserved apical meristems while an enrichment of the culture medium with higher sucrose concentration (90 g l− 1) did not improve regrowth of the apices. Key message: Cryopreservation based on droplet-vitrification technique allows for successful plant regeneration from apical meristem of Tulipa tarda. [ABSTRACT FROM AUTHOR]

Genetic modifications such as mutation and transformation are powerful tools to study the function of genes and proteins in the model liverwort Marchantia polymorpha, but maintaining the resulting germplasm requires a practical, reliable method. Cryopreservation methods allow researchers to maintain mutant and transgenic lines of M. polymorpha. To date, two methods have been developed for cryopreservation of M. polymorpha gemmae: in the first method, unencapsulated gemmae are stored in liquid nitrogen at −­196 °C, and in the second method, encapsulated gemmae are stored in liquid nitrogen at −­196 °C or a deep freezer at −80 °C. In the present study, we developed a simple method named CRUNC (cr yopreservation of un en c apsulated gemmae), which can be used to store unencapsulated, dried gemmae of wild-type and transgenic M. polymorpha lines in liquid nitrogen and in freezers at −80 °C and −20 °C. Using the CRUNC method, we observed a high recovery rate (as high as 100%) and successful long-term (5 months) storage of the gemmae. Therefore, the CRUNC method is practical for maintaining valuable M. polymorpha germplasm.

The history of dermatology reminds a lot of attempts to transform the dense human skin samples into a liquid phase. A search for a method aimed to separate cellular substrate of the skin and to obtain a suspension for studying the cell phenotype profile was a basis of our long-term study, which resulted in a proprietary method for digital estimation of distinct subsets composing the skin cell population. The skin cytoimmunogram is the name of the invention, which is recognized as practical and promising for implementation in public healthcare system as recognosed by the decision of the “Xth International Conference of Immunologists of the Urals”, where the report on the “Prospects for application of the skin cytoimmunogram” was presented for the first time. The results of a single case analysis showed the following findings for this skin sample: 1) a subpopulation of keratinocytes is actively represented, most of them activated, which indicates a moderate proliferative activity of the basal layer of the epidermis; 2) there are B-lymphocytes, which normally are residents of the circulating blood and lymph, since they have a positive taxis to the high endothelial venulas located mainly in the lymph nodes. Their presence in the skin indicates the activity of humoral immunity; 3) presence of several T-cell types of (CD+3 lymphocytes) localized predominantly in the three outer layers of the epidermis, and the findings of CD4+ cells predominating over CD8+ cells suggests an increase in adaptive skin immunity; 4) low content of T-cytotoxic cells indicates to absence of an infectious/inflammatory process; 5) the remaining parameters reflect the numbers of specific skin cells, characterized by low activation grade, which, along with absence of specific complaints in this patient, indicates to normal state of his skin; 6) cell viability in the native sample was 99.8%, after cryopreservation – 87.0%. The proposed method allows to obtain information on the quantitative composition and functional activity of skin cells, which distinctly indicates to the present condition of the patient’s local immunity and may become a basis for development of personalized curative and prophylactic programs. The Сytoimmunogram of skin as a way of skin diagnostic evaluation, is easy to implement, and it is available for qualitative and quantitative evaluation of separate cell subpopulations, in order to assess their function and degree of their response to any external or internal environmental impact. Widespread application of the сytoimmunogram perspective will allow to create sex- and age-matched registry for skin parameters in normal and pathological conditions, thus determining the extent of skin response to environmental exposures, to measure activities of cell subsets in native skin under normal and pathological conditions, to justify the criteria of age-related skin changes, to objectively assess clinical course of skin diseases, and to individually select the drug and monitor the effectiveness of external medical drugs applied.

Annually in Russia about 30 000 cases of oncological diseases in young women are diagnosed. The diagnosis of reproductive problems associated with treatment is still little discussed, although the practical recommendations of many professional organizations, including the American Society of Clinical Oncology, underscore the need for mandatory oncologist review of options for maintaining the patient’s fertility. The article analyzes the medical organization when detected in a female patient of childbearing age cancer is to use therapies aimed at subsequent implementation by the patient of her reproductive rights. It is noted that such opportunities are limited to current law, giving the right to persons with cancer at the expense of budget funds to use the methods of assisted reproduction and cryoconservation technologies for maintaining reproductive potential in cancer patients. The article presents evidence of the urgency of introducing changes to the orders of the Ministry of Health of Russia that limit the use of methods of assisted reproductive technologies for cancer patients at the expense of the budget.

The aim of this study was to examine the effect of the GnRH analogue (buserelin acetate) on the quality of frozen-thawed bovine spermatozoa. In this study, ten Polish Holstein-Friesian bulls with an average age of 3.5 ± 0.5 years were housed individually in pens. From each bull, two ejaculates were collected using an artificial vagina. Semen samples that showed more than 60% motility and 60% viability were selected for this experiment. To the four diluted samples were added 0μg/mL successively; 2μg/mL; 4μg/ mL and 8μg/mL buserelin acetate (Buserelin® aniMedica, Poland). After equilibration, the straws were frozen in liquid nitrogen vapour using a computer controlled automatic freezer. After one day, straws were thawed in a water bath at 38ºC for 20 sec and then were examined. Semen analysis included viability, DNA defragmentation and motility of spermatozoa were being estimated. The addition of 2μg / mL of the GnRH analogue significantly (P <0.05) improved the percentage of live spermatozoa compared to the control group. Samples with supplemented of 2 i 4 μg/mL of buserelin has a significant positive influence on percentage of motile sperm, progressive motility and DNA damage. Improving the quality of frozen semen due to the use of GnRH analogue additive (buserelin acetate) for bovine semen and its low cost of use in routine semen production will allow manufacturers of insemination doses to improve the quality of their product. [ABSTRACT FROM AUTHOR]

Fish populations continue to decline globally, signalling the need for new initiatives to conserve endangered species. Over the past two decades, with advances in our understanding of fish germ line biology, new ex situ management strategies for fish genetics and reproduction have focused on the use of germ line cells. The development of germ cell transplantation techniques for the purposes of propagating fish species, most commonly farmed species such as salmonids, has been gaining interest among conservation scientists as a means of regenerating endangered species. Previously, ex situ conservation methods in fish have been restricted to the cryopreservation of gametes or maintaining captive breeding colonies, both of which face significant challenges that have restricted their widespread implementation. However, advances in germ cell transplantation techniques have made its application in endangered species tangible. Using this approach, it is possible to preserve the genetics of fish species at any stage in their reproductive cycle regardless of sexual maturity or the limitations of brief annual spawning periods. Combining cryopreservation and germ cell transplantation will greatly expand our ability to preserve functional genetic samples from threatened species, to secure fish biodiversity and to produce new individuals to enhance or restore native populations. Australian fish populations are declining with few strategies in place to reintroduce animals if a species were to go extinct. A new technique allows conservationists to freeze and store reproductive tissue from fish indefinitely, and then produce new individuals using a surrogate. This review describes the development of this method and the ways in which it could now be used to protect Australia's vulnerable fish species. [ABSTRACT FROM AUTHOR]

Canine semen cryoconservation was used since 1969, and this process is still nowadays in progress. This review aims to have an overview of two factors leading to a successful freezing–thawing semen. The success and efficiency of freezing process can be measured by the post‐thawing sperm mobility. The first factor is the best extender used as a cryoprotectant to have a similar osmolarity and pH compared to the seminal plasma to enable sperm survival. Historically, chicken egg yolk was used since 1940, but due to microbial risks and to the presence of granules (which interfere with counting dead spermatozoa and inhibits a spermatozoal respiration), despite these disadvantages, egg yolk is considered an excellent cryoprotectant for sperm of different animal species. The low‐density lipoproteins (LDL), contained in EY, when used at a concentration of 6% in a freezing medium associated with 20 mM of glutamine, show a mobility up to 54.5%, which is the best combination found. However, the sperm protection mechanism by LDL during freezing–thawing process only begins to be decrypted. But extraction protocols of LDL are not efficient for an industrial use. Therefore, egg yolk plasma is used within liquid or lyophilized state, and offering similar efficiency as the 6% LDL middle. The equilibration step, in which the diluted sperm is placed for a variable period of time at a temperature of +4°C, before freezing it. The studies show that 6 hr is the optimal duration for the canine sperm equilibration. The future of canine sperm cryopreservation is expected in liposome use and synthetic substances, which mimics LDL role. [ABSTRACT FROM AUTHOR]

The aim of this work was to estimate the effect of vaccinating stallions against anthrax on the sperm quality and its criostability. Vaccination of stallions against anthrax led to a decrease in the sperm quality. During the first week after vaccination, the sperm productivity decreased by 10%. In freshly diluted sperm, the sperm motility decreased by 12%, survival by 38% (Р > 0.99), and membrane integrity by 20% (Р > 0.95) on average. Their recovery was observed not earlier than 28 days after vaccination. Vaccination had an especially strong effect on sperm quality after cryopreservation: sperm motility decreased by 35% (P > 0.99), survival by 45% (P > 0.99), and membrane integrity by 46% (P > 0.99) on average, and these parameters remained low for 1 month. Rejection of sperm doses in the postvaccination period was 50% because of the decrease in quality below the standard level. The amount of pathological types of sperm increased 45 days after vaccination: by 27% (Р > 0.95) in fresh sperm and 34% (Р > 0.99) in deconserved sperm. In the case of cryoconservation of stallions' sperm, it is necessary to take into account the recovery period after antiepizootic measures. [ABSTRACT FROM AUTHOR]

The advances in embryo culture and cryopreservation techniques associated with the potential deleterious effects of ovarian stimulation (SO) on endometrial receptivity and the risk of ovarian hyperstimulation syndrome (SHSO) has allowed the emergence of the "freeze all" strategy. It consists in delaying the transfer of embryos during a cycle without SO and therefore not exposed to supra physiological levels of hormones. Theoretically, this strategy would improve the outcome in terms of efficacy and safety for all patients who undergo OS for IVF/ ICSI. However, according to the recent publications, it appears that, in clinical practice, the strategy of freeze all would be beneficial only for some subgroups of patients: women at risk of OHSS, hyper-responders and candidates for preimplantation genetic diagnosis. In these situations, the freeze all strategy is justified and even recommended. In other situations, freeze all does not seem to bring any improvement in clinical or obstetric or neonatal outcomes. [ABSTRACT FROM AUTHOR]

Contents: The use of artificial insemination in cattle breeding has evolved to global extent, and insemination doses are often shipped via air transport which requires strict radiation‐based examinations. For the determination of effect of non‐ionizing radiation (NIR), to which are beings frequently exposed due to protection of airport or cultural event security, freshly ejaculated and cryopreserved bovine spermatozoa were used as experimental model. Following radiation with hand‐held metal detector in various exposition times (0, 10 s, 15, 30 and 60 min—groups FR, FR10, FR15, FR30 and FR60) the spermatozoa underwent motility and DNA fragmentation analyses. Study on cryoconserved semen treated with NIR was performed in time intervals 0, 10 s, 1 and 5 min (insemination doses radiated before cryoconservation—CB, CB10, CB1, CB5; samples radiated after freezing—CA, CA10, CA1 and CA5). Fresh semen and insemination doses radiated after cryoconservation showed significantly lower total and progressive motility. No effect on motility parameters was detected in semen extended with cryopreservative medium and radiated prior to freezing. Surprisingly, NIR showed a potential to stimulate spermatozoa velocity; however, the effect was modulated throughout the post‐thawing incubation. Based on the DNA fragmentation assay, sperm DNA stayed intact. Present study underlines the potential harm of NIR, which is frequently used in everyday life, with overall adverse impact on human and animal reproduction. Current study also points out on interesting short‐term spermatozoa stimulation induced by NIR. [ABSTRACT FROM AUTHOR]

A significant decrease in the number and genetic diversity of sturgeons is taking place at the current stage. It is not possible to solve the problems concerning the preservation of their biodiversity without the usage of rational approaches and biotech techniques. One of the promising directions for solving this problem is a sperm cryoconservation. However, despite the significant number of studies in this direction, some research issues still remain completely unresolved. For example, the composition of cryoprotective solution used for sperm freezing need to be optimized. The studies, highlighted in this paper, were aimed at finding the ways to optimize the process of sturgeon (namely sterlet) sperm cryoconservation in order to increase the quality of defrosted product and fish-biological results of the fish egg fertilization. The conducted research allowed us to establish the positive results of introduction of a new substances into the cryoprotective solution. The method of the sterlet sperm freezing was also optimized, allowing to improve the investigated parameters. Obtained results can be promising in sturgeon aquaculture, as well as for the sperm and fish egg deposition of rare and endangered fish species in cryobanks. [ABSTRACT FROM AUTHOR]

The article presents a description of a new method of calculation by means of which the distribution of the temperature of a mixture of coolants along the working circuit of a low-temperature refrigerating machine may be calculated to a first approximation and the quantitative composition of a charge mixture of coolants determined for a given composition of the circulating mixture of coolants. [ABSTRACT FROM AUTHOR]

Ex situ collections offer the potential to reduce extinction risks, affording option to society in maintaining future breeding opportunities for productivity and heritage traits. However, how much should we be seeking to collect and conserve in gene banks, and where? We developed a mathematical model to optimize logistical decisions of breed conservation choices and to evaluate alternative scenarios for efficiently re‐allocating genetic materials currently stored in different European gene banks, allowing for cross‐country collections, cost and cryogenic capacity differentials. We show how alternative allocations for the breeds that are currently stored in 11 European gene banks could reduce overall conservation costs by around 20% by selecting cryogenic banks that have relatively lower combination of fixed and collection costs, and are geographically closer to collection regions. Our results show that centralizing collections in one gene bank would double the costs, relative to collective European collections approaches. We also calculate marginal costs of collections and show that increasing diversity within the gene banks implies in higher costs per conserved breed. [ABSTRACT FROM AUTHOR]

Ecuador has a great variety of climatic regions that potentiate biodiversity. The family Orchidaceae constitutes one of the most important of the country, having identified about 4032 species with a high degree of endemism, therefore the development and research of alternative methods of storage and conservation of species is a strategy of primary interest for researchers and for society in general. In cryopreservation, temperatures reach below -190°C in order to paralyze the chemical reactions and keep the plant material viable for long periods. The present research focuses on the development of protocols for cryopreservation of seeds, aimed at the preservation of biodiversity, focusing on the family Orchidaceae, for the subsequent generation of a seed bank. The assays were performed on seeds of Epidendrum quitensium, Sobralia rosea, and Epidendrum anderssonii. Two freezing rates were tested: rapid freezing at -196°C; and step freezing at -22°C, -60°C to 196°C, further analyzed four combinations from Dimethylsulfoxide DMSO, glycerol and sucrose (DMSO 1M; DMSO 1M + glycerol 1M; DMSO 1M + sucrose 1M; DMSO 1M + glycerol 0,5M + sucrose 0,5M). The best results were obtained both in rapid and stepped freezing without the use of cryo-protective substances, by introducing the seeds directly into liquid nitrogen. Species of the genus Epidendrum presented a more efficient response in comparison to Sobralia. The viability of the seeds was evaluated by the tetrazolium test.

Fecal Microbiota Transplantation (FMT) is suggested as an efficacious therapeutic strategy for restoring intestinal microbial balance, and thus for treating disease associated with alteration of gut microbiota. FMT consists of the administration of fresh or frozen fecal microorganisms from a healthy donor into the intestinal tract of diseased patients. At this time, in according to healthcare authorities, FMT is mainly used to treat recurrent Clostridium difficile. Despite the existence of a few existing stool banks worldwide and many studies of the FMT, there is no standard method for producing material for FMT, and there are a multitude of factors that can vary between the institutions. The main constraints for the therapeutic uses of FMT are safety concerns and acceptability. Technical and logistical issues arise when establishing such a non-standardized treatment into clinical practice with safety and proper governance. In this context, our manuscript describes a process of donor safety screening for FMT compiling clinical and biological examinations, questionnaires and interviews of donors. The potential risk of transmission of SARS-CoV-2 virus by the use of fecal microbiota for transplantation must be taken urgently into consideration. We discuss a standardized procedure of collection, preparation and cryopreservation of fecal samples through to the administration of material to patients, and explore the risks and limits of this method of FMT. The future success of medicine employing microbiota transplantation will be tightly related to its modulation and manipulation to combat dysbiosis. To achieve this goal, standard and strict methods need to be established before performing any type of FMT.

Résumé Préserver la fertilité est habituel en cancérologie mais moins systématique au cours des pathologies dysimmunitaires, qui peuvent pourtant justifier l’utilisation de traitements gonadotoxiques chez le sujet jeune, ou affecter la fertilité par elles-mêmes. Cette revue a pour objectif de clarifier les indications et méthodes de préservation de la fertilité dans ce contexte. Le cyclophosphamide est le seul traitement immunosuppresseur nécessitant de préserver la fertilité féminine tandis que chez l’homme, on proposera une préservation avant traitement par cyclophosphamide, méthotrexate, mycophénolate mofétil ou inhibiteurs de mTOR. D’autres facteurs inhérents à la pathologie ou intrinsèques au patient peuvent altérer la fertilité. Pour augmenter les chances de succès, il faut donc anticiper en réalisant un bilan de fertilité, ce qui est possible grâce à un lien permanent entre centre d’assistance médicale à la procréation et interniste. Le choix des modalités de préservation de la fertilité féminine dépendra de l’âge, de l’activité de la maladie, du délai possible avant un traitement, de la possibilité d’une grossesse future mais aussi du désir de la femme voire du couple. Avant la puberté, la seule méthode reconnue est la cryoconservation de tissu ovarien. Chez la femme pubère, la cryoconservation d’ovocytes matures doit être proposée en première intention. En cas d’urgence, de contre-indication ou de refus, on envisagera une cryoconservation de tissu ovarien ou un traitement par agonistes de la GnRH. Pour l’homme après la puberté, on réalisera une cryoconservation de sperme. Avant la puberté, la biopsie testiculaire avec cryoconservation de spermatogonies reste expérimentale. Fertility preservation is routinely performed in cancerology but less systematically used in the field of immune diseases, even though the use of gonadotoxic treatments in young patients may be required and even though the disease itself can alter fertility. This review aimed to clarify the indications and methods of fertility preservation in this context. Cyclophosphamide is the only immunosuppressive drug requiring fertility preservation in women. In men, fertility preservation should be proposed before treatment with cyclophosphamide, methotrexate, mycophenolate mofetil or mTOR inhibitors. Other factors inherent to the disease or the patient may alter fertility. Thus, screening for infertility and fertility preservation have to be implemented as much as possible to increase the chances of successful procreation in patients with immune disease. For women, the choice between the different preservation methods depends on the patient's age, disease activity, the time available before the start of treatment, the possibility of future pregnancy and the woman's and even couple's wishes. Before puberty, the only accepted method is cryopreservation of ovarian tissue. After puberty, the first-line method is the cryopreservation of mature oocytes. If the treatment has to be started in an emergency, if ovarian hyperstimulation/oocyte retrieval is contraindicated or if the patient refuses this option, cryopreservation of ovarian tissue or GnRH agonists could be proposed. For men, the accepted method is sperm cryopreservation. For prepubertal boys, the cryopreservation of spermatogonia after testicular biopsy is still experimental. [ABSTRACT FROM AUTHOR]

Résumé Depuis la parution de la loi de bioéthique de 2004, la préservation de la fertilité fait partie du parcours de soins des patientes devant être traitées par des chimiothérapies et/ou radiothérapie et/ou chirurgie ovarienne gonadotoxiques et des patientes pour lesquelles la fertilité risque d’être prématurément altérée. Il existe plusieurs techniques de préservation de la fertilité féminine, dont trois techniques d’assistance médicale à la procréation : la cryoconservation embryonnaire, d’ovocytes isolés et de cortex ovarien. Le choix de la ou des techniques dépend du type de traitement, des doses et de sa durée, mais aussi de l’âge de la patiente, de son statut marital, de l’urgence de la mise en place de son traitement anticancéreux et de l’hormonosensibilité de la tumeur. Les résultats de ces techniques, dans le cadre de la préservation de la fertilité, sont en pleine évolution et compte tenu d’une utilisation différée des embryons, ovocytes et fragments ovariens cryoconservés, ne sont pas encore définitifs. Since the publication of the Bioethics Act of 2004, the fertility preservation is a part of care of patients that will be treated with gonadotoxic chemotherapy and/or radiotherapy and/or ovarian surgery and of patients with fertility that could be prematurely altered. There are several techniques for preserving female fertility. Three techniques of assisted reproductive biology are possible: embryo cryopreservation, isolated oocytes cryopreservation and ovarian cortex cryopreservation. The choice of the technique(s) depends on the type of treatment, its duration and the doses, but also on the age of the patient, her marital status, the emergency of her anticancer treatment and the hormonosensitivity of the tumor. The results of these techniques of fertility preservation are evolving and given the delayed use of embryos, oocytes and ovarian fragments, are not yet definitive. [ABSTRACT FROM AUTHOR]

Copyright of Revue de Médecine Vétérinaire is the property of Ecole Nationale Veterinaire de Toulouse and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Though cryopreservation of some invertebrate embryos was successful, the cryopreservation of crustacean embryos has not yet been reported. On the basis of previous study, a single vitrifying cocktail (Code A: 30% propanediol and 20% dimethyl formamide) was selected as a cryoprotectant from six kinds of vitrifying solutions to cryopreserve E. sinensis embryos. The cleavage stage, the gastrula, the pre-nauplius, and the first zoea stage embryos were serially acclimated in 25, 33, 50, 67 and 100% vitrifying solution A for 6 min. Embryos were acclimated for 1 min on the 20 cm liquid nitrogen (LN2) layer, and then plunged into liquid nitrogen. After storage for several minutes in LN2, straws containing frozen embryos were acclimated for 1 min on the 20 cm liquid nitrogen (LN2) layer, then quickly removed from LN2. For thawing, straws were immersed quickly for 1 min in a water bath at 37°C, then carefully washed with 0.25 mol/l sucrose and were then incubated with 15 ppt seawater in a Petri dish at 25°C. The survival rate in the vitrifying solution differed for embryos in different stages of development, and the survival rate of different stage embryos declined with the increase of acclimation time in code A vitrifying solution. The survival rate of pre-nauplius stage embryos did not significantly differ when the embryos were washed for 5, 10, 15, or 20 min with 0.25 mol/l sucrose after thawing. There was no survival after either cleavage stage embryos, or gastrula stage embryos were frozen. Eight pre-nauplius stage embryos survived and the survival rate was 9.3 ± 2.5%, but subsequently the embryos died at the fourth day. Seven first zoea stage embryos survived, and the survival rate was 11.3 ± 3.6%, one frozen-thawed embryo hatched at the seventh day. We conclude that cryopreservation of E. sinensis embryos by vitrification is feasible. [ABSTRACT FROM AUTHOR]