Valerate enhances flesh hardness by inhibiting muscle proliferation and differentiation in Nile tilapia (Oreochromis niloticus): An in vivo and in vitro study

Short-chain fatty acids (SCFAs) can enhance the growth of aquatic animals and improve flesh quality through the gut-muscle axis. However, the effects of valeric acid, one of the SCFAs, on aquatic animals remain unreported. This study sought to explore the effects of valeric acid on Nile tilapia ( Oreochromis niloticus ). A total of 360 Juvenile tilapia (mean weight, 1.820 ± 0.007 g) with 3 tanks for each treatment were randomly assigned to 12 rectangular acrylic tanks and fed with four isolipidic (6 % lipid) and isonitrogenous (37 % protein) diets for 60 days, including a control diet (CK) and three experimental diets supplemented with 500 mg/kg (CK + LV), 1000 mg/kg (CK + MV), and 2000 mg/kg (CK + HV) of sodium valerate. The results discovered that the enhanced flesh hardness ( p  < 0.01) of tilapia caused by valerate was attributed to decreased myofiber size ( p  < 0.001). Transcriptome analysis revealed significant enrichment of the Forkhead box O (FOXO) signaling pathway in the valerate-treated group ( p  < 0.001). Subsequent qPCR, western blot, immunohistochemistry, and immunofluorescence analyses demonstrated that valerate inhibited muscle cell proliferation by activating the FOXO signaling pathway, suppressing FOXO nuclear export, and consequently downregulating cyclins and cyclin-dependent kinases (CDKs) ( p  < 0.01). Furthermore, valerate suppressed muscle differentiation by inhibiting the transcription and protein levels expression of myogenic regulatory factors (MRFs) ( p  < 0.001). Additional validation in C2C12 myoblasts and myotubes confirmed that valerate indeed inhibited myoblasts proliferation and myotubes differentiation ( p  < 0.001). These findings indicated that valerate reduced myofiber size by suppressing myoblasts proliferation and myotubes differentiation. This study elucidated the effects and mechanisms of valerate on skeletal muscle and introduced a novel approach to enhance fish flesh hardness.