Antioxidant properties of D-limonene and its nanoemulsion form enhance its anticoccidial efficiency in experimentally infected broilers with Eimeria tenella: an in vitro and in vivo study.

The excessive use of conventional medications to treat coccidiosis has led to concerns regarding drug residues in tissues and the emergence of multidrug resistance. Essential oils with anti-inflammatory and antioxidant activities may also have anticoccidial effects. The present study investigated the efficacy of D-limonene and its nanoemulsion form against Eimeria tenella in chickens. An in vitro study was conducted to evaluate the sporulation inhibitory effects of D-limonene on Eimeria tenella oocysts. Five D-limonene concentrations (0.625, 1.25, 2.5, 5, and 10% v/v) were tested alongside positive (10% formalin) and negative (2.5% potassium dichromate) controls. Each ELISA plate well was inoculated with 1200 unsporulated oocysts and incubated at 30 °C for 24, 48, and 72 h. Subsequently, samples were microscopically examined to assess sporulation inhibition and calculate the percentage of sporulated oocysts. For the in vivo study, 125 eight-day-old broiler chicks were divided into five groups of 25 birds each. The control negative group remained uninfected and untreated. The control positive group was challenged with 5 × 10⁴ sporulated Eimeria tenella oocysts. The diclazuril group received 0.2 mg/kg diclazuril in their diet two days prior to, and until 10 days post infection. The D-limonene (DL) and D-limonene nanoemulsion (DLN) groups were challenged with 5 × 10⁴ sporulated E. tenella oocysts at 18 days of age and administered 150 mg/L of their respective treatments in drinking water from day eight until the end of the experiment. Results from the in vitro study demonstrated that D-limonene suppressed oocyst sporulation by 50.83% at its highest concentration of 10%. In the in vivo study, both DL and DLN treated groups exhibited a significant reduction in oocyst output per gram of feces (OPG), along with increased body weight and decreased parasite stages in the cecal tissue. Furthermore, these treatments were associated with elevated levels of antioxidant enzymes such as glutathione peroxidase (GPX), catalase (CAT), and superoxide dismutase (SOD), accompanied by a decrease in malondialdehyde (MDA) and nitric oxide (NO) levels. Particularly, DLN treatment remarkably increased the number of goblet cells. In conclusion, D-limonene and its nanoemulsion represent promising alternatives for managing coccidiosis in poultry. They not only effectively control parasites but also promote intestinal health and boost antioxidant defenses.