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The establishment of fibroblast lines enables several applications from the formation of biobanks for the conservation of biodiversity to the use of these cells in physiological and toxicological assays. Considered a species vulnerable to extinction, the characterization of fibroblastic lines of northern tiger cat would contribute to its conservation. Therefore, we established and characterized fibroblasts derived from northern tiger cat during extended passage (third, seventh, and eleventh passages) and cryopreservation with regard to the morphology, viability, apoptotic classification, metabolism, proliferative activity, and oxidative stress by reactive oxygen species (ROS) levels and mitochondrial membrane potential (ΔΨm). Initially, we identified four dermal fibroblast lines by morphology, immunophenotyping, and karyotyping assays. In vitro culture after the third, seventh, and eleventh passages did not affect the viability, apoptotic classification, and ROS levels. Nevertheless, cells at seventh and eleventh passages featured a reduction in metabolism and an alteration in ΔΨm when compared to third passage cells. Additionally, cells at eleventh passage showed changes in the proliferative activity and morphology when compared to other passages. Regarding cryopreservation, no effect was observed on cryopreserved cells for morphology, viability, apoptotic classification, metabolism, and proliferative activity. Nevertheless, cryopreserved cells had alteration for ROS levels and ΔΨm. In summary, fibroblasts from northern tiger cat were affected by extended passage (seventh and eleventh passages) and cryopreservation. Adjustments to the in vitro culture and cryopreservation are necessary to reduce cellular oxidative stress caused by in vitro conditions.

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Wastewater-based monitoring has been widely implemented worldwide for the tracking of SARS-CoV-2 outbreaks and other viral diseases. In many surveillance programmes, unprocessed and processed wastewater samples are often frozen and stored for long periods of time in case the identification and tracing of an emerging health threat becomes necessary. However, extensive sample bioarchives may be difficult to maintain due to limitations in ultra-freezer capacity and associated cost. Furthermore, the stability of viruses in such samples has not been systematically investigated and hence the usefulness of bioarchives is unknown. In this study, we assessed the stability of SARS-CoV-2, influenza viruses, noroviruses and the faecal indicator virus, crAssphage, in raw wastewater and purified nucleic aacid extracts stored at -80 °C for 6-24 months. We found that the isolated viral RNA and DNA showed little signs of degradation in storage over 8-24 months, whereas extensive decay viral and loss of qPCR signal was observed during the storage of raw unprocessed wastewater. The most stable viruses were noroviruses and crAssphage, followed by SARS-CoV-2 and influenza A virus. Based on our findings, we conclude that bioarchives comprised of nucleic acid extracts derived from concentrated wastewater samples may be archived long-term, for at least two years, whereas raw wastewater samples may be discarded after one year.

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The combined approaches between ex situ and in situ conservation are of great importance for threatened species in urgent need of protection. This study aims to develop concrete actions to preserve the relic of 30 adult trees of the Sicilian fir (Abies nebrodensis) from extinction using long-term germplasm conservation in liquid nitrogen (LN, -196 °C). Pollen grains were collected, and their moisture content (MC) was measured. Then, viability (2,3,5-tryphenyl tetrazolium chloride, TTC), in vitro germinability, and enzymatic antioxidant activity (ascorbate peroxidase, APX; catalase, CAT) were evaluated before and after cryopreservation. Seeds collected from mature cones underwent X-ray analysis, and only full seeds were used to excise the zygotic embryos (ZEs) for cryopreservation. The MC percentage of ZEs was determined, and then they were plunged in LN with (+PVS2) or without (-PVS2) Plant Vitrification Solution 2; untreated ZEs were used as a control. Viability (TTC test) and in vitro germination were assessed for all ZEs (+PVS2, -PVS2, and control). Embryogenic callus (EC) lines obtained from mature ZEs were cryopreserved applying the 'encapsulation-dehydration' technique. This study has allowed, after optimizing cryopreservation protocols for pollen, ZEs, and EC of A. nebrodensis, to establish the first cryobank of this endangered species in Polizzi Generosa (Palermo, Italy), inside the 'Madonie Regional Park'. The strategy developed for Sicilian fir conservation will pave the way for similar initiatives for other critically endangered conifer species.

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Primordial germ cells (PGCs) constitute an important cell lineage that directly impacts genetic dissemination and species conservation through the creation of cryobanks. In order to advance the field of animal genetic cryopreservation, this work aimed to recover intact PGCs cryopreserved in embryonic tissues during the segmentation phase for subsequent in vitro maintenance, using the yellow-tailed tetra (Astyanax altiparanae) as a model organism. For this, a total of 202 embryos were distributed in two experiments. In the first experiment, embryos in the segmentation phase were dissociated, and isolated PGCs were maintained in vitro. They were visualized using gfp-Pm-ddx4 3'UTR labeling. The second experiment aimed to vitrify PGCs using 3 cryoprotective agents or CPAs (dimethyl sulfoxide, ethylene glycol, and 1,2 propanediol) at 3 molarities (2, 3, and 4 M). The toxicity, somatic cell viability, and recovery of intact PGCs were evaluated. After cryopreservation and thawing, 2 M ethylene glycol produced intact PGCs and somatic cells (44 ± 11.52 % and 42.35 ± 0.33 %, respectively) post-thaw. The recovery of PGCs from frozen embryonic tissues was not possible without the use of CPAs. Thus, the vitrification of PGCs from an important developmental model and Neotropical species such as A. altiparanae was achieved, and the process of isolating and maintaining PGCs in a culture medium was successful. Therefore, to ensure the maintenance of genetic diversity, PGCs obtained during embryonic development in the segmentation phase between 25 and 28 somites were stored through vitrification for future applications in the reconstitution of species through germinal chimerism.

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Plant cryobanks play a significant role in modern science and breeding. They contribute to the recovery of lost species, the emergence of new plant varieties, and help preserve and explore the diversity of the plant world. The IPPRAS Cryobank collection is constantly supplemented with new samples, while, at the same time, the stored samples are being monitored. In order to test seed germination, seeds of Allium and Veratrum species were thawed. Rare Allium species seeds, such as A. nutans, A. schoenoprasum, and A. victorialis were stored in liquid nitrogen for 17, 19, and 30 years, respectively. Long-term cryopreservation decreased germination rates for A. nutans from 96.55 to 50.00%, for A. schoenoprasum from 72.00 to 62.75%, and for A. victorialis from 90.00 to 83.05%. Seeds of a rare medicinal species, Veratrum lobelianum, were stored in liquid nitrogen for 18 years; the seed germination rate during this storage period has been significantly decreased from 75.00 to 14.81%. V. nigrum seeds were also collected and frozen in liquid nitrogen for 3 days. Short-term cryopreservation did not result in a statistically significant change in germination rates (from 79.71 to 82.69%). The seeds of an endangered ornamental species, Cypripedium calceolus, were collected and kept frozen for 3 days. After cryopreservation, the seeds were planted on three different media, as follows: ½ MS, MS with 10% coconut milk, and BM1. On ½ MS medium, 24.98% seeds formed protocorms, while on MS medium with 10% coconut milk, this number was 10.02%, and on BM1 medium, it was 15.02%, respectively; however, after 2.5 months, all of the protocorms died. Thus, it appears that the existing protocol for seed cryopreservation of C. calceolus needs further improvement. The size, weight, and free water content (WC) of six previously cryopreserved Stipa species and three Allium species were measured. For all the Allium and Stipa species studied, we found no correlation between seed size, WC, and cryotolerance. We also found no correlation between the life form, which reflects the water requirement of the species, and cryotolerance.

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Native breed conservation is an important component of poultry biodiversity. The aim of this work is to describe different steps that lead to donor selection for the implementation of the Italian Semen Cryobank of Autochthonous Chicken and Turkey Breeds. The variability within and between breeds was evaluated, and the stored semen reproductive capacity was in vivo tested using artificial insemination. Semen from Bionda Piemontese, Bianca di Saluzzo and Pepoi roosters was collected and processed. Concentration, volume, sperm membrane integrity, total motile sperm, progressive motile sperm and kinetic parameters were analyzed; sperm parameters accounting for bird variability were used to select male donors. Fresh semen quality parameters measured in donor ejaculates showed significant differences between breeds; no differences were found after cryopreservation. Variability in the fertilizing ability of cryopreserved semen was found within a breed (5-16%) and between birds within a breed (BP = 3-7%; BS = 7-31%; PP = 6-22%); only sperm quality parameters measured in fresh ejaculates, not frozen/thawed, may be associated with in vivo fertility results. In conclusion, sperm concentration and progressive motility were successfully used as selection parameters to identify chicken male donors with improved sperm quality for sperm cryobanking. However, new reliable sperm markers to predict cryopreserved semen's fertilizing ability are required.

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Progressive loss of plant diversity requires the protection of wild and agri-/horticultural species. For species whose seeds are extremely short-lived, or rarely or never produce seeds, or whose genetic makeup must be preserved, cryopreservation offers the only possibility for long-term conservation. At temperatures below freezing, most vegetative plant tissues suffer severe damage from ice crystal formation and require protection. In this review, we describe how increasing the concentration of cellular solutes by air drying or adding cryoprotectants, together with rapid cooling, results in a vitrified, highly viscous state in which cells can remain viable and be stored. On this basis, a range of dormant bud-freezing, slow-cooling, and (droplet-)vitrification protocols have been developed, but few are used to cryobank important agricultural/horticultural/timber and threatened species. To improve cryopreservation efficiency, the effects of cryoprotectants and molecular processes need to be understood and the costs for cryobanking reduced. However, overall, the long-term costs of cryopreservation are low, while the benefits are huge.

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Ex situ collections of algae, cyanobacteria, and plant materials (cell cultures, hairy and adventitious root cultures, shoots, etc.) maintained in vitro or in liquid nitrogen (-196 °C, LN) are valuable sources of strains with unique ecological and biotechnological traits. Such collections play a vital role in bioresource conservation, science, and industry development but are rarely covered in publications. Here, we provide an overview of five genetic collections maintained at the Institute of Plant Physiology of the Russian Academy of Sciences (IPPRAS) since the 1950-1970s using in vitro and cryopreservation approaches. These collections represent different levels of plant organization, from individual cells (cell culture collection) to organs (hairy and adventitious root cultures, shoot apices) to in vitro plants. The total collection holdings comprise more than 430 strains of algae and cyanobacteria, over 200 potato clones, 117 cell cultures, and 50 strains of hairy and adventitious root cultures of medicinal and model plant species. The IPPRAS plant cryobank preserves in LN over 1000 specimens of in vitro cultures and seeds of wild and cultivated plants belonging to 457 species and 74 families. Several algae and plant cell culture strains have been adapted for cultivation in bioreactors from laboratory (5-20-L) to pilot (75-L) to semi-industrial (150-630-L) scale for the production of biomass with high nutritive or pharmacological value. Some of the strains with proven biological activities are currently used to produce cosmetics and food supplements. Here, we provide an overview of the current collections' composition and major activities, their use in research, biotechnology, and commercial application. We also highlight the most interesting studies performed with collection strains and discuss strategies for the collections' future development and exploitation in view of current trends in biotechnology and genetic resources conservation.

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During the first outbreak of an emergent virus, methods need to be developed to rapidly establish suitable therapies for patients with high risk of severe disease caused by the pathogen. Considering the importance of the T-cell response in controlling viral infections, adoptive cell therapy with virus-specific T cells has been used as a safe and effective antiviral prophylaxis and treatment for immunocompromised patients. The main objective of this study was to establish an effective and safe method to cryostore whole blood as starting material and to adapt a T-cell activation and expansion protocol to generate an off-the-shelf antiviral therapeutic option. Additionally, we studied how memory T-cell phenotype, clonality based on T-cell receptor, and antigen specificity could condition characteristics of the final expanded T-cell product. Twenty-nine healthy blood donors were selected from a database of convalescent plasma donors with a confirmed history of SARS-CoV-2 infection. Blood was processed using a fully automated, clinical-grade, and 2-step closed system. Eight cryopreserved bags were advanced to the second phase of the protocol to obtain purified mononucleated cells. We adapted the T-cell activation and expansion protocol, without specialized antigen-presenting cells or presenting molecular structures, in a G-Rex culture system with IL-2, IL-7, and IL-15 cytokine stimulation. The adapted protocol successfully activated and expanded virus-specific T cells to generate a T-cell therapeutic product. We observed no major impact of post-symptom onset time of donation on the initial memory T-cell phenotype or clonotypes resulting in minor differences in the final expanded T-cell product. We showed that antigen competition in the expansion of T-cell clones affected the T-cell clonality based on the T-cell receptor ß repertoire. We demonstrated that good manufacturing practice of blood preprocessing and cryopreserving is a successful procedure to obtain an initial cell source able to activate and expand without a specialized antigen-presenting agent. Our 2-step blood processing allowed recruitment of the cell donors independently of the expansion cell protocol timing, facilitating donor, staff, and facility needs. Moreover, the resulting virus-specific T cells could be also banked for further use, notably maintaining viability and antigen specificity after cryopreservation.

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Sperm cryopreservation is an important tool for genetic diversity management programs and the conservation of endangered breeds and species. The most widely used method of sperm conservation is slow freezing, however, during the process, sperm cells suffer from cryoinjury, which reduces their viability and fertility rates. One of the alternatives to slow freezing is vitrification, that consist on rapid freezing, in which viable cells undergo glass-like solidification. This technology requires large concentrations of permeable cryoprotectants (P- CPA's) which increase the viscosity of the medium to prevent intracellular ice formation during cooling and warming, obtaining successful results in vitrification of oocytes and embryos. Unfortunately, this technology failed when applied to vitrification of sperm due to its higher sensitivity to increasing concentrations of P-CPAs. Alternatively, a technique termed 'kinetic sperm vitrification' has been used and consists in a technique of permeant cryoprotectant-free cryopreservation by direct plunging of a sperm suspension into liquid nitrogen. Some of the advantages of kinetic vitrification are the speed of execution and no rate-controlled equipment required. This technique has been used successfully and with better results for motility in human (50-70% motility recovery), dog (42%), fish (82%) and donkey (21.7%). However, more studies are required to improve sperm viability after devitrification, especially when it comes to motility recovery. The objective of this review is to present the principles of kinetic vitrification, the main findings in the literature, and the perspectives for the utilization of this technique as a cryopreservation method.

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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.

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When coral species become extinct, their genetic resources cannot be recovered. Coral cryobanks can be employed to preserve coral samples and thereby maintain the availability of the samples and increase their potential to be restocked. In this study, we developed a procedure to determine coral species-specific requirements for cryobank freezing through determining suitable cryoprotective agents (CPAs), CPA concentrations, equilibration times, holding durations, viability rates, and cell amounts for banked coral cells, and we established the first ever coral cell cryobank. Coral cells, including supporting and gland cells, epidermal nematocysts, Symbiodiniaceae and symbiotic endoderm cells (SEC) were found from the extracted protocol. Approximately half of the corals from the experimental corals consisted of spindle and cluster cells. Gastrodermal nematocysts were the least common. The overall concentration of Symbiodiniaceae in the coral cells was 8.6%. Freezing using DMSO as a CPA was suitable for approximately half of the corals, and for the other half of species, successful cell cryopreservation was achieved using MeOH and EG. EG and DMSO had similar suitabilities for Acanthastrea, Euphyllia, Favites, Lobophyllia, Pavona, Seriatopora, and Turbinaria, as did EG and MeOH for Acropora, Echinopyllia, and Sinularia and MeOH and DMSO for Platygyra after freezing. At least 14 straws from each species of coral were cryobanked in this study, totaling more than 1884 straws (0.5 mL) with an average concentration of 6.4 × 106 per mL. The results of this study may serve as a framework for cryobanks worldwide and contribute to the long-term conservation of coral reefs.

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RESEARCH QUESTION: Is the efficacy of imported vitrified oocyte donation affected by the cryobank of origin? DESIGN: Longitudinal cohort study, including 249 completed oocyte warming cycles from 200 recipients (January 2016-July 2020). No severe male factor was included. Vitrified oocytes were provided by three Spanish cryobanks. Primary outcome was cumulative live birth delivery rate (CLBR) per completed oocyte warming cycle. RESULTS: After warming 1535 oocytes, 1244 survived (81.0%) and 945 fertilized (76.0%); embryo utilization rate was 65.3%. The overall CLBR per completed cycle was 47.0% but was lower in cryobank 1 (31.2%) versus cryobank 2 (56.0%, P = 0.0010) and cryobank 3 (50.8%, P = 0.0241). Multivariate logistic regression analysis identified survival of four or more oocytes as the strongest predictor for delivery (P = 0.0282). Only 202 out of 249 oocyte warming cycles had four or more survived oocytes in a proportion that was significantly lower in cryobank 1 versus cryobank 2 (70.1% versus 89.0%, P = 0.0020); comparison with cryobank 3 (81.0%) was not significant. In the 202 oocyte warming cycles, CLBR in cryobank 1 (37.0%) was lower versus cryobank 2 (58.8%, P = 0.0115) and cryobank 3 (60.8%, P = 0.0019), suggesting a reduced viability in oocytes from cryobank 1 that survived warming. CONCLUSIONS: Differences were found in the efficacy of imported vitrified oocytes in relation to the cryobank of origin. Each centre needs to evaluate the results internally when starting a collaboration with an oocyte cryobank to establish the necessary measures to maximize treatment efficacy.

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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.

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Cryopreservation is currently the only method which allows long-term conservation of living clonal plant material in the vapor or liquid phase of nitrogen (at -140 to -196 °C) allowing tissue to be viable for decades or perhaps centuries. Specifically, for species with recalcitrant seeds or requiring constant vegetative propagation, it is the method of choice for the long-term conservation of its genetic resources. The protocol described here is a modification of a previously developed plant vitrification solution 2 (PVS2)-droplet vitrification method of potato shoot tips, adapted from Musa species. Utilizing this protocol, the International Potato Center (CIP) has successfully stored in the cryobank more than 3000 cultivated potato accessions, belonging to seven species and nine different taxa [16], originating principally from ten countries in South and Central America. As part of CIP's quality management system, all vegetative material placed in cryo is routinely subsampled, thawed, and assessed to confirm that whole plantlets can be produced after storage in liquid nitrogen. Complete plant recovery rates of thawed shoot tips range from 20% to 100% (average rate: 60%). This chapter describes the complete set of steps from the routine procedure of cryopreserving potato shoot tips for long-term conservation.

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Buffalo is an important farm animal species in South and South-east Asian countries. Cryopreservation allows long-term storage of somatic cells, which can be made available to research communities. This study aimed to 1) establish and cryopreserve somatic cells from elite buffaloes, and 2) share stored somatic cells and their associated data with researchers. To achieve these targets, somatic cells were established successfully from tail-skin biopsies of 17 buffaloes. The informative data such as buffalo details (breed, date of birth, sex, and age at the time of tissue biopsy collection, and production traits), the number of cryovials stored, and freezing dates were recorded in an electronic file and a printed inventory record. The established somatic cells were flat, spindle-shaped morphology, and expressed vimentin (a fibroblast-like cell type marker) and the negative expression of cytokeratin-18 (an epithelial cell type marker). Altogether, we cryopreserved 970 cryovials (0.1 million cells per vial) from two buffalo breeds, namely Murrah and Nili-Ravi (at least 45 cryovials per animal), for cryobanking. Somatic cell nuclear transfer (SCNT) experiments demonstrated the utility of cryopreserved cells to produce cloned buffaloes. Importantly, these cryopreserved somatic cells are made available to scientific communities. This study encourages the cryopreservation of somatic cells of elite farm animals for their utilization in cell-based research.

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PURPOSE: To evaluate the effectiveness, efficiency, and safety of a transnational gamete donation (TGD) programme based on the shipping of vitrified donor oocytes. METHODS: A retro-prospective observational study was conducted in the Assisted Reproductive Technology Center of the University Hospital of Florence, Italy. The study population included 622 consecutive donor oocyte cycles. A mean number of 6 vitrified oocytes per couple were shipped from two Spanish biobanks. In the receiving centre, gametes were warmed and inseminated and the subsequent embryo transfer (ET) was performed. The main outcome measurement was LBR. Secondary outcomes included oocyte survival rate, ICSI damage rate, normal fertilization, cleavage, and implantation rate (IR) in both 'fresh' and cryotransfer cycles. RESULTS: A total number of 3536 mature oocytes were warmed with 81.4% surviving oocytes. 2PN oocytes were 1941 with an ICSI normal fertilization rate of 70.4% and a cleavage rate of 93.4%; 857 day-3 embryos were transferred in 498 women, 63 blastocysts in 44. Couples with at least one vitrified embryo were 181 (32.3%). IR was 25.1% and 33.1% for day-3 ET and blastocyst stage respectively. Crude pregnancy rate and LBR after the first ET were 35.5% and 27% correspondingly with a conservative cumulative LBR of 34% and an optimal LBR of 51.4%. CONCLUSION: Imported vitrified donor oocytes retain their competence and are capable of resulting in ongoing pregnancies and healthy babies in a proportion comparable to other existing systems as egg donation with vitrification/warming in the same laboratory and transnational fresh oocyte donation.

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STUDY QUESTION: Which is the most suitable clinical strategy in egg donation IVF cycles conducted with imported donated vitrified oocytes? SUMMARY ANSWER: The importation, and allocation, of at least eight vitrified eggs per couple during an egg donation cycle is associated with a high cumulative live birth delivery rate per cycle, as well as the confident adoption of a single blastocyst transfer strategy to minimize the risk of multiple pregnancies. WHAT IS KNOWN ALREADY: IVF using donor eggs is commonly used worldwide to treat women who are unable to conceive with their own oocytes. In 2014, the Constitutional Court (n.162/2014) gave permission for gamete donation to be allowed for ART in Italy. Initially recommended as a therapeutic approach for premature ovarian insufficiency, the use of donated oocytes has become more and more common. In countries such as Italy, fresh oocyte donation is theoretically possible, but practically impossible due to the lack of donors. In fact, the Italian law does not allow reimbursement to the young women, who can only voluntarily donate their eggs. Therefore, Italian IVF centers have established several collaborations with international oocyte cryo-banks. The most popular workflow involves the importation of donated oocytes that have been vitrified. However, recent evidence has questioned the overall efficacy of such an approach. This is because detrimental effects arising from oocyte vitrification and warming might reduce the number of eggs available for insemination, with a consequential reduction in the achievable live birth rate per cycle. STUDY DESIGN, SIZE, DURATION: This was a longitudinal cohort study, conducted between October 2015 and December 2018 at two private IVF centers. Overall, 273 couples were treated (mean maternal age: 42.5 ± 3.5 years, range: 31-50 years; mean donor age: 25.7 ± 4.2, 20-35 years) with oocytes purchased from three different Spanish egg banks. PARTICIPANTS/MATERIALS, SETTING, METHODS: We performed an overall analysis, as well as several sub-analyses clustering the data according to the year of treatment (2015-2016, 2017 or 2018), the number of warmed (6, 7, 8 or 9) and surviving oocytes (≤4, 5, 6, 7, 8 or 9) and the cycle strategy adopted (cleavage stage embryo transfer and vitrification, cleavage stage embryo transfer and blastocyst vitrification, blastocyst stage embryo transfer and vitrification). This study aimed to create a workflow to maximize IVF efficacy, efficiency, and safety, during egg donation cycles with imported vitrified oocytes. The primary outcome was the cumulative live birth delivery rate among completed cycles (i.e. cycles where at least a delivery of a live birth was achieved, or no embryo was produced/left to transfer). All cycles, along with their embryological, obstetric and neonatal outcomes, were registered and inspected. MAIN RESULTS AND THE ROLE OF CHANCE: The survival rate after warming was 86 ± 16%. When 6, 7, 8 and 9 oocytes were warmed, 94, 100, 72 and 70% of cycles were completed, resulting in 35, 44, 69 and 59% cumulative live birth delivery rates per completed cycle, respectively. When ≤4, 5, 6, 7, 8 and 9 oocytes survived, 98, 94, 85, 84, 66 and 68% of cycles were completed, resulting in 16, 46, 50, 61, 76 and 60% cumulative live birth delivery rates per completed cycle, respectively. When correcting for donor age, and oocyte bank, in a multivariate logistic regression analysis, warming eight to nine oocytes resulted in an odds ratio (OR) of 2.5 (95% CI: 1.07-6.03, P = 0.03) for the cumulative live birth delivery rate per completed cycle with respect to six to seven oocytes. Similarly, when seven to nine oocytes survived warming, the OR was 2.7 (95% CI: 1.28-5.71, P < 0.01) with respect to ≤6 oocytes. When cleavage stage embryos were transferred, a single embryo transfer strategy was adopted in 17% of cases (N = 28/162); the live birth delivery rate per transfer was 26% (n = 43/162), but among the pregnancies to term, 28% involved twins (n = 12/43). Conversely, when blastocysts were transferred, a single embryo transfer strategy was adopted in 96% of cases (n = 224/234) with a 30% live birth delivery rate per transfer (N = 70/234), and the pregnancies to term were all singleton (n = 70/70). During the study period, 125 babies were born from 113 patients. When comparing the obstetric outcomes for the cleavage and blastocyst stage transfer strategies, the only significant difference was the prevalence of low birthweight: 34 versus 5%, respectively (P < 0.01). However, several significant differences were identified when comparing singleton with twin pregnancies; in fact, the latter resulted in a generally lower birthweight (mean ± SD: 3048 ± 566 g versus 2271 ± 247 g, P < 0.01), a significantly shorter gestation (38 ± 2 versus 36 ± 2 weeks, P < 0.01), solely Caesarean sections (72 versus 100%, P = 0.02), a higher prevalence of low birthweight (8 versus 86%, P < 0.01), small newborns for gestational age (24 versus 57%, P = 0.02) and preterm births (25 versus 86%, P < 0.01). LIMITATIONS, REASONS FOR CAUTION: This retrospective study should now be confirmed across several IVF centers and with a greater sample size in order to improve the accuracy of the sub-analyses. WIDER IMPLICATIONS OF THE FINDINGS: Single blastocyst transfer is the most suitable approach to achieve high success rates per procedure, thereby also limiting the obstetric complications that arise from twin pregnancies in oocyte donation programs. In this regard, the larger the cohort of imported donated vitrified oocytes, the more efficient the management of each cycle. STUDY FUNDING/COMPETING INTEREST(S): None. TRIAL REGISTRATION NUMBER: None.

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The present study aimed to find an effective cryopreservation protocol for turkey semen through the combined use of dimethylsulfoxide (DMSO) and three non-permeant cryoprotectants (NP-CPAs), sucrose, trehalose, and Ficoll 70. In addition, the action of two dilution rates (1:2 and 1:4) were also investigated. Semen was processed according to two final dilution rates and the following treatments: Tselutin extender (TE)/DMSO (control), TE/DMSO + sucrose or trehalose 50, 100, 200, or 400 mM, and TE/DMSO + Ficoll 0.5, 0.75, 1, or 1.5 mM. In total 26 different combinations treatments were achieved. The diluted semen was filled up into straws and frozen on liquid nitrogen vapor. The post-thawing sperm quality was assessed by analyzing motility, membrane integrity, osmotic resistance, and DNA integrity. The results obtained revealed a significant effect of NP-CPA concentration on total and progressive motility, on most of the kinetic parameters, on membrane integrity and DNA integrity, while the post-thaw quality was less affected by dilution rate. The highest post-thaw quality for all sperm quality parameters assessed except curvilinear velocity (VCL) and DNA integrity were found in semen frozen with 1 mM Ficoll/1:4 (p < 0.05). Our findings provide an important contribution for the identification of a reference procedure for turkey semen cryopreservation, in order to create the first national avian semen cryobank.

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The aim of our study was to test the effectiveness of a simple semen cryopreservation procedure, developed for cultivated salmonid, on the wild salmonid of the Mediterranean area and to evaluate the effect of different thawing rates and sperm-to-egg ratios. The semen of five individual males was diluted into a final extender concentration of 0.15 M glucose and 7.5% methanol and loaded into 0.25 mL plastic straws, and a final sperm concentration of 3.0 × 109 sperm/mL was obtained. After equilibration, the straws were frozen by exposure to liquid nitrogen vapor at 3 cm above the liquid nitrogen level for 5 min. The semen was thawed at 40 °C/5 s or 10 °C/30 s. The sperm cryosurvival was evaluated by examining in vitro the sperm motility parameters using the CASA system, followed by fertilization trials in vivo, using three different sperm-to-egg ratios 6 × 105, 4.5 × 105 and 3 × 105:1. The applied cryopreservation procedure resulted in remarkably high (85.6%) post-thaw sperm total motility, when the semen was thawed at 40 °C/5 s, whilst the highest fertilization rate (53.1%) was recorded for a sperm-to-egg ratio of 4.5 × 105:1. According to these outcomes, the cryopreservation procedure that was tested turned out to be effective for the wild population of Mediterranean brown trout and practical for the creation of the first European semen cryobank foreseen as part of our "LIFE" Nat.Sal.Mo. project.