Genome Integrity in Dairy Cows Fed Black Soldier Fly Oil: An Integrated Sister Chromatid Exchange and Alkaline Comet In Vivo Assessment

Background/Objectives: Insect-derived lipids are emerging as circular-economy feed in-gredients, but their implementation in ruminant diets requires robust safety assessment beyond productive endpoints. This study evaluated genome integrity in 26 lactating Val-dostana Red Pied cows fed concentrates containing either hydrogenated palm fat (HPF; n = 13) or black soldier fly oil (Hermetia illucens oil, HIO; n = 13) for 50 days. Methods: Pe-ripheral blood lymphocytes were analyzed using Sister Chromatid Exchanges (SCE), re-flecting replication-associated chromosomal instability, and the alkaline Comet assay, quantifying primary DNA damage at the single-cell level (Tail DNA and Olive tail mo-ment) at T0 (the day before the start of the two experimental diets), T1 (30 d) and T2 (50 d). Results: Baseline SCE frequencies were comparable between groups. Over time, SCE values decreased in both groups, but a significant reduction occurred only in HIO at day 50, with lower SCE frequency than HPF (5.73 ± 0.11 vs. 6.29 ± 0.13; p = 0.002). Comet tail DNA showed a significant time effect (T0 vs. T1: mean difference = 179,846.6; p < 0.001; T0 vs. T2: mean difference = 138,395.2; p = 0.012), with diet-dependent modulation. In fact, in HIO, tail DNA decreased from 387,886 ± 94,606 (T0) to 147,006 ± 30,592 (T1; p < 0.001), remained lower at day 50 (155,723 ± 29,357; p = 0.024), and was lower than HPF at both T1 (p = 0.006) and T2 (p = 0.009). Olive tail moment also decreased over time (T0 vs. T1: mean difference = 1.925 × 1015; p = 0.008; T0 vs. T2: mean difference = 1.676 × 1015; p = 0.025), and it differed between diets at day 50 in favor of HIO (5.99 × 1015 ± 5.45 × 1014 vs. 7.26 × 1015 ± 5.98 × 1014; p = 0.017). Conclusions: Overall, no evidence of genotoxicity was observed in cows fed HIO; conversely, the results support compatibility with genome stability and suggest a modest time-dependent improvement detectable mainly after prolonged sup-plementation.