Non-lethal screening of relative omega-3 status in live fish using ocular near-infrared hyperspectral imaging

Rapid, non-lethal screening of omega-3 status could support selective breeding and quality monitoring in aquaculture. We tested the eye as an accessible site for near-infrared hyperspectral imaging (NIR-HSI; 869–1771 nm) in juvenile largemouth bass (Micropterus salmoides). Ocular spectra were paired with free-fatty-acid reference measurements for 259 fish from five dietary treatments and 20 tanks. Low, Middle and High categories were defined by the 33rd and 67th percentiles of the training fish in each validation fold. In four-fold nested tank-grouped validation, each outer fold held out one complete tank per diet, and the model assigned every fish to one of the three classes. EPA achieved an accuracy of 0.625, balanced accuracy of 0.620 and macro-F1 of 0.604; corresponding values were 0.552, 0.556 and 0.521 for EPA/DHA and 0.405, 0.402 and 0.401 for DHA. High-class recall was 0.909 for EPA and 0.894 for EPA/DHA. The final models were frozen and applied without refitting to 20 fish from a separately reared batch that was not used in model development. Independent-batch accuracies were 0.70 for EPA, 0.75 for DHA and 0.70 for EPA/DHA; High-class recalls were 1.00, 1.00 and 0.90, respectively. Performance was strongest and most consistent for the High class. These results support ocular NIR-HSI as a non-lethal prioritization tool for relative free-fatty-acid screening in live fish.