Grape Pomace Extract Restores Antioxidant Capacity in Nosema ceranae-Infected Honey Bees

Nosema ceranae, intracellular microsporidian parasite, is among the most prevalent and damaging pathogens of the honey bee (Apis mellifera), inducing chronic oxidative stress and impairing the antioxidant defense capacity of infected workers. In this study, we investigated the potential of a hydroethanolic extract of Prokupac grape pomace (Vitis vinifera L.), known for its rich polyphenolic composition and strong in vitro antioxidant activity, to modulate oxidative stress in N. ceranae-infected bees in in vivo conditions. A cage experiment was performed and oxidative stress was studied on days 7 and 14 of the experiment by means of L-band electron paramagnetic resonance (EPR) spectroscopy using TEMPOL as a spin probe, with faster signal decay indicating greater antioxidant and reducing activity. Infection with N. ceranae significantly suppressed TEMPOL reduction compared to healthy controls, confirming infection-induced oxidative impairment that increased over time. In contrast, uninfected bees receiving the extract showed the highest reduction rates, exceeding even the negative control, demonstrating that the extract actively enhances antioxidant capacity in healthy bees. Supplementation with the extract also improved the survival of infected bees and reduced N. ceranae spore loads, while food consumption did not differ among the groups. These results indicate that Prokupac grape pomace extract has significant in vivo antioxidant effects in honey bees, and that the timing of supplementation appears to influence the degree of antioxidant protection in infected bees, highlighting its potential as a sustainable, naturally derived dietary supplement for bee health management.