Integrated In Silico Modeling and In Vivo Trials to Investigate the Functional Role of Nigella sativa Seeds as a Feed Additive for Enhancing Performance, Physiology, and Stress Resilience in Heat-Stressed Farâfra Lambs

Background and Objective: Heat stress (HS) is one of the stressors that impairs various physiological and immunological conditions in an animal’s body. Small ruminants have a principal role in the economy. Lambs are young sheep under one year and have a huge nutritional and economic value. Nigella sativa (NS) is a perennial growing plant from the Ranunculaceae family. Both NS seeds (NSS) and NS oil have a wide range of therapeutic properties as immunomodulators, antimicrobials, and anti-inflammatories. This research aims to assess the functional potential of NSS as a natural nutritional supplement to enhance growth, antioxidant defense, immune function, and physiological resilience in heat-stressed Farâfra lambs to preserve the livestock. Materials and Methods: NS compounds were explored through computational molecular docking, where seventeen bioactive ligands were screened against 473 Ovis aries protein structures from the AlphaFold database to identify key immune-related molecular interactions. After that, an in vivo study was performed using eighteen Farâfra lambs (7-8 months old, 38 ± 2.56 kg), which were randomly allocated to three groups: control (0 g NSS/day), NS-S6 (6 g NSS/day), and NS-S12 (12 g NSS/day), with six lambs per group. The feeding trial lasted 60 days following a 7-day adaptation period. Growth performance, blood biochemical indices, and physiological variables, including rectal temperature, skin temperature, breathing rate, and heart rate, were measured throughout the experiment. Results: Molecular docking analysis showed strong binding of alpha-longipinene to the melatonin receptor 1A, suggesting possible immune-enhancing activity, while Frizzled-4 was identified as another key target involved in Wnt-mediated immune regulation. Experimentally, NSS supplementation improved average daily gain, feed efficiency, antioxidant capacity, and immune parameters while stabilizing physiological responses to HS. Conclusion: Supplementation with NSS enhanced growth performance, immunity, and stress tolerance in Farâfra lambs exposed to HS. The combination of in silico and in vivo findings highlights the immunomodulatory and antioxidant potential of NSS, supporting its use as a natural, functional feed additive to encourage livestock resilience and productivity under thermal stress.