Therapeutic potential of calabrian Citrus essential oils as sustainable anthelmintics against Haemonchus contortus and Trichostrongylus colubriformis in sheep: In vitro assessment

Gastrointestinal nematodes (GINs), particularly Haemonchus contortus and Trichostrongylus colubriformis, threaten sheep health and welfare globally, while anthelmintic resistance drives the need for sustainable alternatives. Essential oils (EOs) from Citrus species, rich in bioactive terpenes, represent a promising option, especially as locally sourced by-products. Five furocoumarin-free (FCF) Citrus EOs from Calabria (bergamot, sweet orange, lemon, mandarin, grapefruit) were chemically characterised by GC-MS. Anthelmintic activity against H. contortus and T. colubriformis was evaluated using the egg hatch test (EHT) at six concentrations (0.049–50 mg/mL). IC50 values were calculated by non-linear regression or linear interpolation. Egg hatch inhibition percentages were compared using two-way ANOVA with essential oil and concentration as fixed factors, followed by Tukey's multiple-comparison test (p < 0.05). Limonene dominated all EOs (25.17–47.85%); bergamot EO contained higher linalyl acetate (13.50%) and linalool (11.32%). All EOs showed concentration-dependent inhibitory effects on egg hatching. At 0.781 mg/mL, lemon (IC50 = 0.62 mg/mL) and mandarin (IC50 < 0.78 mg/mL) were the most potent against H. contortus, followed by orange (0.95 mg/mL), bergamot (1.69 mg/mL), and grapefruit (1.87 mg/mL). Against T. colubriformis, orange was remarkably potent (IC50 < 0.195 mg/mL). Maximum activity ranged from 82.7% to 90.7%. Two-way ANOVA revealed highly significant effects of concentration, EO identity, and their interaction (p < 0.001). A strong inter-species correlation in EO activity was observed (r = 0.938; p < 0.05). This study provides the first systematic evidence for in vitro anthelmintic activity of five Calabrian Citrus EOs against key stages of H. contortus and T. colubriformis. Lemon and mandarin EOs are the most potent candidates in the egg hatch test. Their multi-component composition offers a theoretical advantage in terms of a multi-target mechanism, which may be relevant for resistance management, although this hypothesis requires experimental validation. These findings suggest that Calabrian Citrus EOs warrant further investigation as locally sourced, sustainable anthelmintic candidates for integrated parasite management in sheep, particularly in organic and low-input farming systems. However, in vivo validation is essential before any practical recommendations can be made.