Studies on methods of triploidy percentage analysis. In：C.S. Lee, M.S. Su and I C. Liao (eds.)

Abstract

For the induction of triploidy in a variety of aquatic organisms, thermal shock, hydrostatic pressure shock and chemical shock have been applied to inhibit second polar body formation in fish and the first or second polar body formation in shellfish. For the identification of triploidy, microscope cytophotometry, flow cytometry and chromosome preparation were recommended although measurement of erythrocyte and nuclear volume and silver staining were two other methods ever studied. Cytophotometric DNA assay was based on the quantity of light absorbed by the stained nuclei and was expressed as the absorption unit. Blood smears of 20-day old fry were made on slides and fixed in methanol : formalin : 9 : 1: v/v for 15 min. Slides were washed twice with distilled water, dried and then stained using the Feulgen reaction procedure. DNA content of individual erythrocyte nuclei was measured and computed with a Leitz MPV 3 microscope photometer. In the preparation of fish chromosome, gill, embryo or small fish was treated with hypotonic solution, that is, either 0.005N potassium chloride, 0.005 N sodium citrite or distilled water. The resulting material was then fixed with Carnoy solution. The tissues were finely chopped using a scalpel. The cell suspension was spread on a warm slide to form a cell ring. Proper staining was accomplished by using Giemsa solution. When flow cytometry was applied,the red blood cells were treated with 0.2 ml saturated EDTA solution and PBS-BSA-NaN3 solution up to 1 ml. The mixture was kept at 4°C before analysis and was then centrifuged at 1,100 rpm for 5 min. After the supernatent was discarded, the cells were treated with 0.3 ml solution A (0.5 ml RNase + 0.1 ml Triton-X-100 + 0.5 ml Propidium iodide) and incubated at 37°C for 20 min. It was then treated with 0.3 ml solution B (0.5 ml Propidium iodide + 0.1 ml Triton X-100 + 9.4 ml 0.4 M NaC1) and incubated at 4°C for one hour. The samples were protected from light and the flow cytometric analysis was processed allowing the sample injection at optimum flow rate, sheath pressure and laser power to obtain better HPCV (half pick coefficient of variation) and histograms. The histograms were analyzed using the supplied DNA analysis program and a recommended software. The three methods used in triploidy identification have their own particular characteristics. In instruments used, microscope cytophotometry (MC) , makes use of the fluorescent microscope; chromosome preparation (CP), the light microscope; and flow cytometry (FC), the flow cytometer. In the target material to be studied, MC and FC aim for the DNA content in the red blood cell while CP, the chromosome number in the cellular nuclei of the embryo, gill or cell line. In the possible errors when using the different methods, MC has focusing and condenser errors; CP, overlapping of and missing chromosomes during preparation; and FC, poor pretreatment and inconsistent laser power. In the number of tested cells per individual finfish or shellfish, MC has 30 ± 10 cells; CP, 30-100 cells; and FC, > >10,000 cells. In pretreatment time per individual, MC requires 40 min; CP, 3-12 h; and FC, 3 min. Lastly, in either observation or sample run time per individual, it takes about 1 h for MC; 1 h for CP; and 2 min for FC.