Docosahexaenoic acid (DHA) promotes grass carp (Ctenopharyngodon idella) muscle fiber development by activating MEK/ERK pathway in vitro and in vivo

Abstract

Docosahexaenoic acid (DHA) is an essential nutrient for aquatic animals and plays a key role in promoting the growth performance of fish. This study was designed to evaluate the effect of DHA on the development of grass carp muscle fiber in vitro and in vivo. In vitro, myoblasts isolated from grass carp were incubated with 50 μM DHA for 24 h. EDU (5-ethynyl-2′ -deoxyuridine) fluorescence staining showed that 50 μM DHA increased the proportion of EDU-positive cells (P < 0.05). And flow cytometry analysis indicated 50 μM DHA also significantly increased the ratio of S and G2 phase cells and decreased the ratio of G1 phase cells (P < 0.05). In addition, expression analysis of proliferation-related genes further showed that 50 μM DHA promoted the proliferation of myoblasts (P < 0.05). Subsequent transcriptome analysis found that the MEK/ERK signaling pathway was activated during this process. Furthermore, the MEK inhibitor trametinib (Tra) inhibited the proliferative effect of DHA on myoblasts, confirming the causal involvement of this pathway. In vivo, 270 fish (initial weight: 27.92 ± 0.02 g) were randomly fed one of the 3 diets for 8 weeks (each diet had 3 replicates): control diet (Con), 1% DHA diet (1% DHA) and the diet supplement with both 1% DHA and 50 mg/kg trametinib (DHA + Tra). The final body weight, specific growth rate, and protein efficiency were significantly increased in the 1% DHA group (P < 0.05). No significant differences were found in crude protein, ash, crude lipid, and moisture levels of the whole body among the 3 groups, whereas the crude protein content in muscle was significantly increased in the 1% DHA group (P < 0.05), suggesting that the increase of FBW was due to muscle growth. Importantly, the fiber density and sarcomere lengths in the 1% DHA group was significantly higher than those of the control and 1% DHA + Tra groups (P < 0.05). The principal component analysis (PCA) on muscle amino acid and fatty acid well separated the DHA group from the control group, and the total weight rank of the muscle amino acid in DHA group appeared to be optimal. The mRNA expression of MEK, ERK and genes related to muscle development significantly increased in the 1% DHA group compared to the control and 1% DHA + Tra groups (P < 0.05). Similarly, the protein expression of myosin heavy chain (MyHc), myoblast determination protein (Myod) and myogenin (Myog) significantly increased in the 1% DHA group. Together, these results suggest that DHA promotes the muscle fiber development likely via the activation of MEK/ERK pathway in grass carp.