Effects of dietary isoleucine level on growth and expression of genes related to nutritional and physiological metabolism of swimming crab (Portunus trituberculatus)

A six-week feeding trial was conducted to evaluate the effects of dietary isoleucine level on growth, myofiber histology, and mRNA levels of genes related to amino acid, glucose and lipid metabolism in swimming crab (Portunus trituberculatus). Six isonitrogenous and isolipidic diets (45% protein and 8% lipid) were formulated to contain graded levels of isoleucine (Ile), with the analyzed levels of isoleucine being 0.69%, 1.06%, 1.32%, 1.78%, 2.03% and 2.38%, respectively. A total of 180 crabs (initial weight 16.30 ± 0.01 g) were housed individually in 180 plastic baskets with 30 crabs assigned to each diet, divided randomly into three replicates of 10 crabs. Each crab was fed twice at 08:00 and 18:00 with a daily feeding ration of 6–8% of crab body weight. Eight crabs from each replicate group were sampled and dissected after the feeding trial to provide hemolymph for biochemical analysis, hepatopancreas for proximate composition, amino acid profile enzyme activity, and gene expression, and muscle for proximate composition, amino acid profile and histology. The results indicated that final weight, percent weight gain and specific growth rate increased significantly as dietary isoleucine level increased from 0.69% to 1.78%, and then decreased with further increased dietary isoleucine. Crabs fed the diets with 1.78%, 2.03% and 2.38% isoleucine showed significantly higher muscle protein contents (17.80%, 17.99% and 17.65% of wet mass) than crabs fed lower levels of isoleucine. The highest concentrations of IGF1R and CHH in hemolymph (187.95 and 529.42 pg ml−1, respectively) and hepatopancreas (24.18 and 38.69 pg ml−1, respectively) were observed in crabs fed the diet with 1.32% isoleucine. Hepatopancreas expression levels of genes related to amino acid metabolism, including s6k1, bcat, alt and ast, were up-regulated in crabs fed 1.32% isoleucine compared to the other groups, whereas the expression level of tor decreased as dietary isoleucine increased. Expression levels of genes related to glucose intake, such as pi3k, igf1r and glut1, were significantly higher in crabs fed 1.32% isoleucine compared to crabs fed 0.69% isoleucine. Expression levels of genes related to gluconeogenesis (pepck and g6pase) were significantly higher in crabs fed 0.69% isoleucine than in crabs fed the diets with 1.32% and 1.78% isoleucine. In contrast, expression levels of genes related to glycolysis, including hk, gk and pk, were up-regulated in crabs fed 1.32% isoleucine compared to crabs fed 0.69% isoleucine. Gene expression levels of regulatory factors related to lipid metabolism (foxo, sirt1 and sirt3) were significantly higher in crabs fed 1.32% isoleucine compared to crabs fed the other treatments, while expression levels of genes related to lipolysis (hsl and cpt2) were significantly up-regulated in crabs fed the diets with 1.32% and 1.78% isoleucine. Expression levels of key lipogenesis genes (fas and acc) were down-regulated in crabs fed the diets with 1.32% and 1.78% isoleucine compared to those fed 0.69% isoleucine. Muscle histology indicated that crabs fed the diets with 1.32% and 1.78% isoleucine had significantly lower densities of myofibers (21.67 and 17.33 N/ mm2) compared to crabs fed 0.69% isoleucine. In conclusion, based on percent weight gain, the optimal isoleucine requirement of juvenile swimming crab was calculated to be 1.57% of diet (3.49% dietary protein) by quadratic curve model. Thus, appropriate dietary isoleucine levels (1.32–1.78%) promoted amino acid metabolism and protein synthesis, increased protein deposition, and altered myofiber structure of muscle and, in addition, promoted lipolysis and glycolysis, and reduced lipogenesis and gluconeogenesis in juvenile swimming crab.