The supplementation of a microdiet with crystalline indispensable amino-acids affects muscle growth and the expression pattern of related genes in Senegalese sole (Solea senegalensis) larvae

Abstract

The full expression of growth potential in fish larvae largely depends on an efficient protein utilization, which requires that all the indispensable amino acids (IAAs) are provided at an optimum ratio. The effect of supplementing a practical microdiet with encapsulated crystalline-AA to correct possible IAA deficiencies was evaluated in Senegalese sole larvae. Two isonitrogenous and isoenergetic microdiets were formulated and processed to have approximately the same ingredients and proximate composition. The control diet (CTRL) was based on protein sources commonly used in the aquafeed industry. In the supplemented diet (SUP) 8% of an encapsulated fish protein hydrolysate was replaced by crystalline-AA in order to increase the dietary IAA levels. The microdiets were delivered from mouth-opening upon a co-feeding regime until 51days after hatching (DAH). The larvae capacity to utilize protein was evaluated using an in vivo method of controlled tube-feeding during relevant stages throughout development: pre-metamorphosis (13DAH); metamorphosis climax (19DAH) and metamorphosis completion (25DAH). Somatic growth was monitored during the whole trial. A possible effect on the regulation of muscle growth was evaluated through muscle cellularity and the expression of related genes (myf5, myod2, myogenin, mrf4, myhc and mstn1) at metamorphosis climax (19DAH) and at a juvenile stage (51DAH). The SUP diet had a negative impact on larvae somatic growth after the metamorphosis, even though it had no effect on the development of Senegalese sole larvae capacity to retain protein. Instead, changes in somatic growth may reflect alterations on muscle growth regulation, since muscle cellularity suggested delayed muscle development in the SUP group at 51DAH. Transcript levels of key genes regulating myogenesis changed between groups, during the metamorphosis climax and at the 51DAH. The group fed the SUP diet had lower dnmt3b mRNA levels compared to the CTRL group. Further studies are needed to ascertain whether this would possibly lead to an overall DNA hypomethylation in skeletal muscle. Statement of relevanceIn farmed fish species, there has been a great effort over the years to provide the best conditions for successful development of embryos and small larvae, as early environmental conditions can strongly affect muscle growth during early stages and influence the subsequent growth potential at later life stages. In spite of increased efforts to understand the regulation of myogenesis by intrinsic factors like genotype and extrinsic factors such as photoperiod and temperature, studies evaluating the impact of nutritional factors on fish larvae muscle development are still very scarce.This work will raise interest to the discussion on whether a nutritional cue during an early developmental stage can impact on the regulation of muscle growth and on further growth potential in a metamorphosing farmed fish species, such as Senegalese sole. Supplementing microdiets with crystalline-AA in order to correct dietary IAA did not improve Senegalese sole larvae somatic growth and led to changes on the regulation of muscle growth associated with changes in expression patterns of muscle growth markers during the trial (secondary MRFs myogenin and mrf4, myhc and mstn1). Dietary IAA affected the expression of a DNA methyltransferase essential for de novo methylation, dnmt3b, suggesting that an epigenetic effect at the transcriptional regulation level may explain differences found in somatic growth as a response to a nutritional cue.This work may contribute to lay down basis for future studies on nutritional programming of muscle growth in fish larvae of important farmed species.