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Can Flow Cytometric DNA Content Analysis Become a Routine Procedure in Aquaculture?

FLOW cytometry (FCM) or laser scanning cytometry (LSC) applied with an appropriate DNA-specific fluorescent dye can serve as a sensitive technique for quantifying the ploidy level of cells (1-3). Other methods such as the determination of chromosome counts are more labor intensive and require the presence of actively dividing cells (4). FCM is widely used in medical diagnostic applications for detection of aneuploid cells or apoptotic cells with fractional DNA content (5). Ploidy level determination is frequently applied in the evaluation of breast, prostatic, or bladder carcinomas. Ploidy level is also an important prognostic factor of hematolymphoid malignancies (e.g., B-lymphoblastic leukemia and plasma cell myeloma) (6). On the other hand, flow cytometric DNA content analysis is often used in taxonomical as well as in evolutional studies to determine the ploidy level of plants (7, 8) and various animal species including insects (9), fishes (10, 11), and mammals. In addition to research applications, ploidy level determination has an increasing significance in plant and animal breeding and in aquaculture. Triploid fishes are generally sterile, because the irregular meiotic segregation of chromosomes results in reduced gonadal development and abnormal gametogenesis (12, 13). In aquaculture industry, it is of special interest to achieve sterile triploid individuals, because they spend less energy on reproductive processes and thus they have a potential for improved growth and longevity in case of many fish species (12). Interestingly, triploid fishes may have different glycogen or fat content also affecting their nutritional value and taste. Of note, generation of sterile triploid individuals may prevent the spreading of foreign species outside culture facilities and thus may avoid the genetic and/or ecological impact of escapees on the natural fish populations (13). Therefore, sterile triploids of foreign fish species can be released into natural water bodies for aquatic weed management without the danger of overpopulation or the establishment and dispersion of unwanted species (13). Unfortunately triploidy induction treatments are not 100% efficient, thus methods to quantify the proportion of triploid larvae generated by a particular treatment can help researchers to select appropriate treatments for efficient triploid induction in a cost-effective manner (14). Although flow cytometric DNA content analysis is a sensitive, reliable, and high-throughput technique, it has several pitfalls. For instance, inappropriate sample preparation or storage conditions may induce disintegration or aggregation of cells leading to false results and/or low measurability of samples. The usage of membrane impermeable DNA-specific dyes requires permeabilization of cells. However, the choice of reagents used for permeabilization and/or fixation of cells may also affect the stoichiometric staining of nuclear DNA (2, 15). Unfortunately, in the practice of fisheries, it is not always possible to obtain fresh samples for immediate analysis. In addition, experimental animals are frequently sampled far from the laboratory resulting in a significant transportation time. Therefore, there is a great demand for sample fixation and storage methods that can be carried out easily even at field conditions and do not worsen the quality of samples making possible their analysis at later time points. Martin Hubálek and Martin Flajšhans (published in the current issue of Cytometry Part A (page XXX)) tested the applicability of 13 simple extended storage methods on sterlet and tench belonging to Chondrostei and Teleostei, respectively. Different tissue specimens were studied including blood and fin tissue of sub-adult/adult individuals and tail tissue of hatched larvae. After fixation procedures, samples were stored for 1, 5, or 10 days and then stained with 40,6-diamidine-20-phenylindole dihydrochloride (DAPI) and Department of Biophysics and Cell Biology, Faculty of Medicine, University of Debrecen, Debrecen, Egyetem tér 1, H-4032, Hungary