The Effect of Ultrasound-Treated Donkey Milk on some Serum Antioxidant and Cytokine Levels as well as ANAE-Positive Lymphocyte Ratios in Rat Trial Models

This study aimed to compare the effects of slow pasteurization and ultrasound treatment on the physicochemical and microbiological quality of donkey milk (DM) and to investigate how these processing methods influence specific antioxidant and immunological responses in rats. In the first stage, donkey milk samples were collected from twenty-two healthy donkeys and categorized into three groups: raw (Rm), pasteurized (Pst; 65 °C for 30 min), and ultrasound-treated (Ult; 100% amplitude for 6 min). Physicochemical parameters (e.g., pH, color, water activity, titratable acidity) and nutritional composition (dry matter, protein, fat, lactose) were determined. Microbial analyses included total aerobic mesophilic bacteria, coliforms, molds/yeasts, coagulase-positive Staphylococcus, and lactic acid bacteria counts. In the second stage, twenty-eight adult male Wistar albino rats were assigned to four groups (control, Rm, Pst, Ult) and each experimental group received 48 mL of the respective DM daily for four weeks. At the end of the feeding period, the rats were sacrificed, and blood samples were collected. Serum levels of malondialdehyde (MDA), glutathione (GSH), catalase (CAT), superoxide dismutase (SOD), and various cytokines (IL-1β, TNF-α, IL-6, IL-10) were quantified. Differential leukocyte counts (lymphocytes, monocytes, neutrophils) and ANAE-positive lymphocyte percentages were also assessed. Although MDA and GSH levels were unaffected by DM type, SOD activities were significantly higher in all DM-fed groups relative to controls. Ultrasound-treated DM showed a more pronounced impact on pro-inflammatory cytokine levels (IL-1β, TNF-α, IL-6) than pasteurized milk, while IL-10 remained unchanged among the groups. Neither pasteurization nor ultrasound treatment affected the differential WBC or ANAE-positive lymphocyte percentages. Overall, ultrasound treatment offered a superior microbial load reduction without adversely impacting antioxidant or bioactive components. These findings suggest that ultrasound application may serve as a viable alternative to traditional pasteurization for preserving the functional properties of donkey milk.