Sampling conditions affect immune cell viability and function in the Mediterranean mussel (Mytilus galloprovincialis)

Farmed animal welfare is becoming a socially relevant issue. Several strategies have been developed to assess the welfare of farmed animals in vitro, including the evaluation of immune functions. The appropriate isolation of immune cells plays a key role in studying in vitro cell functions. This study aims to evaluate the best sampling conditions to promote the viability and immune functions of Mediterranean mussel (Mytilus galloprovincialis) hemocytes. Mussels were divided into 4 groups, based on the solution used for sampling: Modified Leibovitz's 15 medium (mL15), Lithium Heparin + mL15 (LiH + mL15), Modified Alsever's Solution (MAS), and Filtered SeaWater (FSW). Hemolymph was collected, and the total hemocyte count (THC), membrane-glycoprotein retention, viability, and phagocytosis were examined. The MAS group showed significantly higher THC compared to the other groups, while the highest glycoprotein retention was observed in LiH + mL15. In vitro incubation time did not influence hemocyte viability, but hemocytes sampled with LiH + mL15 showed higher viability compared to the MAS and FSW groups. The LiH + mL15 group also displayed greater phagocytic capacity. The results indicate that MAS is recommended for the THC, while, for functional studies, it is suggested to use LiH + mL15, as it best preserves hemocyte viability, membrane glycoproteins, and phagocytic activity and capacity.