Liver Kinase B1 (LKB1) is a serine/threonine kinase that can regulate energy metabolism and skeletal muscle growth. In the present study, LKB1 cDNA of triploid crucian carp (Carassius auratus) was cloned. The cDNA contains a complete open reading frame (ORF), with a length of 1326 bp, encoding 442 amino acids. Phylogenetic tree analysis showed that the LKB1 amino acid sequence of the triploid crucian carp had a high sequence similarity and identity with carp (Cyprinus carpio). Tissue expression analysis revealed that LKB1 was widely expressed in various tissues. LKB1 expressions in the brain were highest, followed by kidney and muscle. In the short-term LKB1 activator and inhibitor injection experiment, when LKB1 was activated for 72 h, expressions of myogenic differentiation (MyoD), muscle regulatory factor (MRF4), myogenic factor (MyoG) and myostatin 1 (MSTN1) were markedly elevated and the content of inosine monophosphate (IMP) in muscle was significantly increased. When LKB1 was inhibited for 72 h, expressions of MyoD, MyoG, MRF4 and MSTN1 were markedly decreased. The long-term injection experiment of the LKB1 activator revealed that, when LKB1 was activated for 15 days, its muscle fibers were significantly larger and tighter than the control group. In texture profile analysis, it showed smaller hardness and adhesion, greater elasticity and chewiness. Contrastingly, when LKB1 was inhibited for 9 days, its muscle fibers were significantly smaller, while the gap between muscle fibers was significantly larger. Texture profile analysis showed that adhesion was significantly higher than the control group. A feeding trial on triploid crucian carp showed that with dietary lysine-glutamate dipeptide concentration increasing, the expression of the LKB1 gene gradually increased and was highest when dipeptide concentration was 1.6%. These findings may provide new insights into the effects of LKB1 on fish skeletal muscle growth and muscle quality, and will provide a potential application value in improvement of aquaculture feed formula.

AlaSerCys Transporter 2 (ASCT2), encoded by the SLC1A5 gene, plays an important role in the absorption of glutamine. In this study, the full-length cDNA sequence of ASCT2 was cloned from triploid crucian carp. It encodes 539 amino acid residues and a stop codon. Phylogenetic analysis revealed that the sequences of the ASCT2 ORF region in cyprinid fishes shared high sequence homology. Comparing the abundance of ASCT2 in different tissues, we found its expression level in muscle was significantly higher than that in intestine (p < 0.05). In addition, the expression levels of ASCT2 also appeared different in diurnal variation. Then we found the addition of 2.5% glutamate in a feeding diet significantly increased the expression levels of ASCT2 in intestine and muscle (p < 0.05). However, in glutamine experiments, the muscle showed the highest expression level of ASCT2 when fish were fed the diet containing 3.0% glutamine (p < 0.05). In vitro, ASCT2 was sensitive to glutamine and its expression level appeared down-regulated when the addition of glutamine was added to 0.1 mg/mL. Finally, we found that the diet with 29% protein level significantly increased the expression level of ASCT2 in intestine (p < 0.05). Nevertheless, different protein sources (fish meal and soybean meal) had no significant effect on the expression levels of ASCT2 in intestine and muscle (p > 0.05). These results provided data for the study of ASCT2 in triploid crucian carp regulated by feeding nutrition, which had a potential application in improving feed formulation in aquaculture.

Methionine (Met) and lysine (Lys), as primary limiting amino acids, play important roles in regulating muscle quality in aquatic animals. This study investigated the effects of dietary Met and Lys supplementation on the growth performance, antioxidant function, and muscle quality of Hefang crucian carp (HCC) using physiological and transcriptomic analyses. Fish were fed three diets for 8 weeks: a basal diet (LA) and two diets supplemented with DL-methionine at 1.7% (MA) and 3.4% (HA), respectively, while L-lysine supplementation was fixed at 3.4%. The results indicated that dietary Met and Lys supplementation had no significant effect on the growth performance of HCC (p > 0.05), but significantly reduced serum triglyceride (TG) and cholesterol levels (T-CHO) (p < 0.05). Additionally, serum total protein (TP) content was significantly increased in the MA group (p < 0.05). Analysis of serum antioxidant enzyme activities indicated that appropriate Met and Lys supplementation improved antioxidant capacity and upregulated the expression of antioxidant-related genes (Nrf2, GPX1a, GSTO1, GSTP1) in the muscle. Moreover, the MA group exhibited superior muscle hardness and gumminess, while the HA group had higher springiness and chewiness (p < 0.05). Muscle fiber density was significantly increased, whereas diameter and area showed opposite trends in fish fed 1.7% Met and 3.4% Lys (p < 0.05). Furthermore, appropriate Met and Lys supplementation significantly affected muscle fiber development genes (MyoD, MyoG, and MRF4), with MyoG highest in the HA group and MyoD and MRF4 highest in the MA group (p < 0.05). Comparative transcriptomic analysis of muscle tissue showed DEGs were mainly enriched in pathways correlated with muscle quality, involving cardiac muscle contraction, actin cytoskeleton regulation, PPAR signaling pathway and ECM–receptor interaction. Taken together, our findings enhance the understanding of the molecular mechanisms underlying the effects of dietary Met and Lys on muscle quality in HCC, providing valuable insights for the development of nutritional strategies in the aquaculture industry.