This review is focused on the applications of genome cryobanking of aquatic species including freshwater and marine fish, as well as invertebrates. It also reviews the latest advances in cryobanking of model species, widely used by the scientific community worldwide, because of their applications in several fields. The state of the art of cryopreservation of different cellular types (sperm, oocytes, embryos, somatic cells and primordial germ cells or early spermatogonia) is discussed focusing on the advantages and disadvantages of each procedure according to different applications. A special review on the need of standardization of protocols has also been carried out. In summary, this comprehensive review provides information on the practical details of applications of genome cryobanking in a range of aquatic species worldwide, including the cryobanks established in Europe, USA, Brazil, Australia and New Zealand, the species and type of cells that constitute these banks and the utilization of the samples preserved. Statement of relevance This review compiles the last advances on germplasm cryobanking of freshwater and marine fish species and invertebrates, with high value for commercial aquaculture or conservation. It is reviewed the most promising cryopreservation protocols for different cell types, embryos and larvae that could be applied in programs for genetic improvement, broodstock management or conservation of stocks to guarantee culture production.
Flow cytometry can be used to obtain high-resolution estimates of nuclear DNA content (8,12,27). Much of the work in this area has been confined to clinical studies in the human, but the technology has been extended to organisms such as pocket gophers (241, triploid trout ( 2 3 , side-necked turtles (61, sex-reversed horses (16), and oysters (1).In these and similar studies, the DNA content of target cells is quantified relative to a standard DNA content in cells from a reference species (13,14,28). Standards may be used as internal references, when target cells and reference cells are mixed together and assayed simultaneously, or as external references, when target cells and cells from the reference species are analyzed independently. When external references are used, the flow cytometer must be checked for equilibration between analyses.The standard can be used for several calculations. The standard can be assigned a known DNA mass, against which picogram quantities of nuclear DNA from target cells can be estimated directly (15). Alternatively, a standard could be used as an internal reference in the analysis of individual samples, following which the reference is cancelled during estimation of a DNA Index. An internal reference would also be cancelled during the calculation of picogram quantities of nuclear DNA relative to a separate standard that has a known DNA mass (and a known relationship to the internal reference). These last two calculations do not require knowledge of the precise DNA mass of the internal reference.Because the use of reference standards provides a relative measure of DNA content, it is crucial that the reference cells and the target cells have a similar DNA content to minimize possible zero shift error (28) and, in the case of a n internal reference, that the values for the reference and target cells do not overlap.In order to gather information relating to the selection of reference standards for use in flow cytometry, we have surveyed a wide spectrum of taxa representing each of the major vertebrate classes. Here we present the results of our survey in 45 species. These species were selected because of their broad distribution and general accessibility, and because they provide an array of fluorescence peaks spanning the range of DNA masses that might be encountered in comparative studies. ~~~
Advancing the production efficiency and profitability of aquaculture is dependent upon the ability to utilize a diverse array of genetic resources. The ultimate goals of aquaculture genomics, genetics and breeding research are to enhance aquaculture production efficiency, sustainability, product quality, and profitability in support of the commercial sector and for the benefit of consumers. In order to achieve these goals, it is important to understand the genomic structure and organization of aquaculture species, and their genomic and phenomic variations, as well as the genetic basis of traits and their interrelationships. In addition, it is also important to understand the mechanisms of regulation and evolutionary conservation at the levels of genome, transcriptome, proteome, epigenome, and systems biology. With genomic information and information between the genomes and phenomes, technologies for marker/causal mutation-assisted selection, genome selection, and genome editing can be developed for applications in aquaculture. A set of genomic tools and resources must be made available including reference genome sequences and their annotations (including coding and non-coding regulatory elements), genome-wide polymorphic markers, efficient genotyping platforms, high-density and high-resolution linkage maps, and transcriptome resources including non-coding transcripts. Genomic and genetic control of important performance and production traits, such as disease resistance, feed conversion efficiency, growth rate, processing yield, behaviour, reproductive characteristics, and tolerance to environmental stressors like low dissolved oxygen, high or low water temperature and salinity, must be understood. QTL need to be identified, validated across strains, lines and populations, and their mechanisms of control understood. Causal gene(s) need to be identified. Genetic and epigenetic regulation of important aquaculture traits need to be determined, and technologies for marker-assisted selection, causal gene/mutation-assisted selection, genome selection, and genome editing using CRISPR and other technologies must be developed, demonstrated with applicability, and application to aquaculture industries.Major progress has been made in aquaculture genomics for dozens of fish and shellfish species including the development of genetic linkage maps, physical maps, microarrays, single nucleotide polymorphism (SNP) arrays, transcriptome databases and various stages of genome reference sequences. This paper provides a general review of the current status, challenges and future research needs of aquaculture genomics, genetics, and breeding, with a focus on major aquaculture species in the United States: catfish, rainbow trout, Atlantic salmon, tilapia, striped bass, oysters, and shrimp. While the overall research priorities and the practical goals are similar across various aquaculture species, the current status in each species should dictate the next priority areas within the species. This paper is an output of the USDA Workshop fo...
Sperm cryopreservation offers potential for long-term storage of genetic resources. However, the current protocols for zebrafish Danio rerio are cumbersome and poorly reproducible. Our objective was to facilitate adoption of cryopreservation by streamlining methods from sperm collection through thawing and use. First, sperm activation was evaluated, and motility was completely inhibited when osmolality of the extender was ≥ 295 to 300 mOsmol/kg. To evaluate cryoprotectant toxicity, sperm were incubated with dimethyl sulfoxide (DMSO), N, N-dimethyl acetamide (DMA), methanol, or glycerol at 5, 10, and 15% concentrations. Based on motility, DMSO, DMA, and methanol (≤ 10%) were less toxic; therefore, sperm were cryopreserved using these cryoprotectants at cooling rates of 10 and 20 °C/min. The highest motility (mean ± SD) (35 ± 23%; P ≤ 0.0001) and fertility (13 ± 8%; P ≤ 0.001) in thawed sperm were obtained with the combination of 8% methanol and a cooling rate of 10 °C/min. Further evaluations of 8% methanol and 10 °C/min were performed with males from populations with high (2.05 ± 0.24) and low (1.18 ± 0.12) body condition (P = 0.0001). Motility of thawed sperm from the two populations was 38 ± 16% and 78 ± 10% (P = 0.0001), and fertilization was 6 ± 6% and 33 ± 20% (P = 0.0001). These values were positively related with body condition factor. Overall, this study simplified and standardized sperm cryopreservation, and established a protocol using French straws as a freezing container and an extender without powdered milk. This protocol can be readily adapted for high-throughput application using automated equipment, and motility and fertility comparable to previous reports were obtained. Male variability and sperm quality remain important considerations for future work, especially in mutant and inbred lines.
We measured genome size (nuclear DNA content) by fluorescence flow cytometry in 55 species of birds representing 12 different orders. Similar studies were performed in approximately 100 species by laboratories using absorption cytophotometry of Feulgen-stained nuclei. Although there have been apparent discrepancies in the assigned values for the species used as a reference, the values obtained in the different laboratories are generally in agreement. When the data are standardized in relation to a diploid (2C) value of 2.5 picograms (pg) of DNA for the domestic chicken (Gallus gallus domesticus), the mean for DNA content in 135 species representing 17 orders is 2.82 +/- 0.33 (SD) pg with a range of 2.0-3.8 pg. Thus the genome size of birds is the most conservative of any vertebrate class and, all values considered, is smaller and more uniform in size than previous estimates would indicate. This could be explained by a previously unexplored hypothesis: that the genome of birds has evolved from a small ancestral genome that was reduced before emergence of the protoavian.
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