In vitro study of a dose- and time- effect response of a combination of organic acids and nature-identical compounds on gilthead seabream head-kidney leucocytes

Organic acids (OA) and botanicals have a long history of use as feed additives in animal production with the aim of improving intestinal health and performance. To the best of our knowledge, the available studies in aquaculture are very limited and little is known about their impact on the cells of the immune system. Aim of this study was to assess the biological properties of OA and nature-identical compounds (NIC) on head-kidney leucocytes (HKLs) viability, functionality and oxidative response in an in vitro model. HKLs were isolated from gilthead seabream (Sparus aurata) and incubated with a combination of citric and sorbic acid, thymol and vanillin (MIX) at 0, 50, 100, 250, 500, and 1000 mg/L for 30 min, 2 h and 4 h. At any assayed time, the MIX did not affect leucocyte viability (which was determined by flow cytometry using propidium iodide) nor phagocytic ability. Regarding respiratory burst, starting at 30 min the MIX at 50 and 100 mg/L induced a decrease in activity compared to control.

The relative gene expression of HKLs after being incubated for 30 min, 2 hour and 4 hours without (control) or with different concentrations of the MIX was also studied by real time PCR. The selected antioxidant genes were nuclear factor erythroid 2 (nrf-2), superoxide dismutase (sod), catalase (cat) and glutathione reductase (gr). The expression of interleukin 1-beta (Il-1 beta) and interleukins 6, 7, 8, 15 and 18 (Il-6, 7, 8, 15, 18) as pro-inflammatory genes and interleukin 10 (Il-10) and transforming growth factor β (tgf-β) as anti-inflammatory genes was also determined. Different significant variations were detected in the expression of all the studied genes on HKLs after being incubated with the different concentrations of the MIX. The increases or decreases recorded were related to the MIX concentration and to the incubation time.

In conclusion, the present results showed that the MIX of OA and NIC did not have a negative impact on HKLs viability and the decrease in the respiratory burst activity in a dose-dependent manner underlines the potential antioxidant power of the MIX. The variation in the expression of antioxidant genes and interleukins suggests a possible modulation of the immune-response mediated by the MIX that needs to be further evaluated in vivo.

Acknowledgements: This research was funded by Vetagro S.p.A. and POR-FESR 2014-2020 Emilia Romagna grant.

For further information: technicalservice@vetagro.com