A protocol for ploidy determination in ethanol-fixed Atlantic salmon (Salmo salar) fry using propidium iodide and...

Ploidy verification in salmonid production is often constrained by the need to analyze fresh samples. Here, we present an optimized protocol for early-stage ploidy determination in Atlantic salmon (Salmo salar) fry using 70 % ethanol fixation at -20 °C and flow cytometry. A staining solution containing Propidium Iodide (PI) and RNase A was employed to ensure stoichiometric DNA quantification, combined with internal biological standardization to minimize instrumental variation. The protocol achieved a 92.7 % analytical measurability rate and a mean coefficient of variation (CV) of 3.10 %, comparable to values reported for fresh blood samples. The normalized fluorescence ratio discriminated all valid samples, yielding values of 1.00 ± 0.05 for diploids (2n) and 1.38 ± 0.04 for triploids (3n) produced by thermal shock applied at 15 and 30 min post-fertilization. Cross-validation by nuclear morphometry confirmed a significant increase in the major nuclear diameter of putative triploids (7.69 ± 1.04 µm) relative to diploids (6.63 ± 0.83 µm). However, an area under the curve (AUC) of 0.76 indicated lower diagnostic performance than flow cytometry. This protocol provides a practical method for ploidy assessment in Atlantic salmon fry, compatible with field conditions at the sample preservation stage.