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 ingredients, but their implementation in ruminant diets requires robust safety assessment beyond productive endpoints. This study evaluated genome integrity in 26 lactating Valdostana 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: Peripheral blood lymphocytes were analyzed using Sister Chromatid Exchanges (SCE), reflecting replication-associated chromosomal instability, and the alkaline Comet assay, quantifying primary DNA damage at the single-cell level (Tail DNA and Olive tail moment) 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 supplementation.