Abstract
Background:
Lead (Pb) is a heavy metal that impairs organ function and immune competence in teleost fish.

Aim:
This study explored the protective effect of dietary milk thistle extract (Silybum marianum; MT) as a natural feed additive against sub-lethal lead acetate-induced toxicity in common carp (Cyprinus carpio L.).

Methods:
A total of 120 fish with a starting weight of 18.5 ± 0.5 g were randomly allocated into four triplicate groups over a 60-day feeding trial: an unexposed control group (CT), a lead acetate-exposed group (LA; 7 mg/L), a milk thistle supplemented group (MT; 5 g/kg diet), and an MT-supplemented group co-exposed to lead acetate (MT + LA). Various parameters, including liver and kidney function biomarkers, antioxidant enzymes, and gill, hepatopancreas, and intestine histopathology, were analyzed across all experimental groups.

Results:
Lead exposure significantly elevated serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) activities, as well as urea, creatinine, and uric acid concentrations, while significantly reducing serum lysozyme and myeloperoxidase (MPO) activities and albumin, globulin, and total protein concentrations relative to CT (P < 0.05). The hepatic superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and total antioxidant capacity (T-AOC) activities were significantly decreased, and the malondialdehyde (MDA) concentration was significantly elevated in the LA group. Histopathological examination of the gills, hepatopancreas, and intestine of Pb-exposed fish revealed lamellar necrosis in the gills, hepatocellular necrosis with vacuolar degeneration in the hepatopancreas, and necrosis of the villus tip in the intestine. Dietary MT supplementation significantly mitigated Pb-induced hepato-renal dysfunction, immunosuppression, and hypoproteinemia, while significantly enhancing hepatic antioxidant enzyme activities and reducing lipid peroxidation. Hepatic enzyme activities remained partially elevated in the MT + LA group, and reductions in gill lamellar necrosis, hepatic lytic necrosis, and intestinal villus necrosis scores did not reach statistical significance relative to the LA group; however, hepatic vacuolar degeneration was significantly ameliorated. Fish supplemented with MT alone did not differ significantly from CT in most parameters and exhibited enhanced innate immune and antioxidant responses.

Conclusion:
Dietary supplementation with 5 g/kg of milk thistle extract partially improved lead-induced hepato-renal dysfunction, oxidative stress, immunosuppression, and histopathological lesions in common carp, which contributed to the use of natural substitutes as immunostimulants and antioxidants.

Key words: Antioxidant enzymes; Cyprinus carpio; Milk thistle extract; Oxidative stress; Silybum marianum.