A protocol for ploidy determination in ethanol-fixed Atlantic salmon (

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.