LAUSR

Abstract Immune stimulators are essential to the development of the aquaculture industry. Cinnamaldehyde (CA) and Bacillus spp. are promising immunostimulatory feed additives, as demonstrated by their beneficial effects in several fish species. This study investigated the effects of dietary CA and Bacillus subtilis either alone or in combination on the growth, digestive enzyme activity, and non-specific immunity of tongue sole, Cynoglossus semilaevis. A total of 240C. semilaevis (188 ± 5 g) were randomly assigned to four dietary treatments. Diets containing no additives (the control group, D), 0.1 g/100 g CA (Dcin), 107 CFU/g B. subtilis (DBac), and CA combined with B. subtilis (Dcin+Bac) were used to feed fish for 60 days. The results revealed significant improvements in the weight gain, specific growth rate, feed conversion ratio, and protein efficiency in the fish fed diets containing either CA or B. subtilis, and there were more significant improvements in the group with the combined supplementation (Dcin+Bac) compared with the control group. Fish fed diets with CA or CA combined with B. subtilis had higher intestinal proteinase, amylase, and lipase activities than the control fish. Dietary CA and B. subtilis singularly or in combination improved the antioxidant capacity and the immune status by stimulating SOD, CAT, and T-AOC activities and GSH-Px levels, by reducing malondialdehyde (MDA) content, and by increasing lysozyme activity in the serum, liver, mid kidney, and spleen. The results of the two-way analysis of variance revealed the synergistic effects of CA and B. subtilis in improving feed conversion ratio, protease activities in the foregut and midgut, and part of the antioxidant parameters. In addition, significant shifts in the overall intestinal microbial community and diversity were analyzed using high-throughput sequencing of the V3/V4 region of the 16S rRNA gene. The results showed that both dietary supplementation of CA alone or in combination with B. subtilis increased the abundance of the probiotic bacterial genera Bacillus, Lactobacillus, and Bifidobacterium. In conclusion, dietary supplementation of cinnamaldehyde and B. subtilis both solely and in combination are beneficial for improving the growth performance, digestive ability, non-specific immunity, and intestinal microbiota community of the tongue sole, Cynoglossus semilaevis.