Dietary protein levels changed the hardness of muscle by acting on muscle fiber growth and the metabolism of collagen in sub-adult grass carp (Ctenopharyngodon idella)

Abstract

Background: Nutrient regulation has been proven to be an effective way to improve the flesh quality in fish. As a necessary nutrient for fish growth, protein accounts for the highest proportion in the fish diet and is expensive. Although our team found that the effect of protein on the muscle hardness of grass carp was probably related to an increased collagen content, the mechanism for this effect has not been deeply explored. Moreover, few studies have explored the protein requirements of sub-adult grass crap (Ctenopharyngodon idella). Therefore, the effects of different dietary protein levels on the growth performance, nutritional value, muscle hardness, muscle fiber growth, collagen metabolism and related molecule expression in grass carp were investigated.

Methods: A total of 450 healthy grass carp (721.16 ± 1.98 g) were selected and assigned randomly to six experimental groups with three replicates each (n = 25/replicate), and were fed six diets with 15.91%, 19.39%, 22.10%, 25.59%, 28.53% and 31.42% protein for 60 d.

Results: Appropriate levels of dietary protein increased the feed intake, percentage weight gain, specific growth rate, body composition, unsaturated fatty acid content in muscle, partial free amino acid content in muscle, and muscle hardness of grass carp. These protein levels also increased the muscle fiber density, the frequency of new muscle fibers, the contents of collagen and IGF-1, and the enzyme activities of prolyl 4-hydroxylases and lysyloxidase, and decreased the activity of matrix metalloproteinase-2. At the molecular level, the optimal dietary protein increased collagen type I α1 (Colα1), Colα2, PI3K, Akt, S6K1, La ribonucleoprotein domain family member 6a (LARP6a), TGF-β1, Smad2, Smad4, Smad3, tissue inhibitor of metalloproteinase-2, MyoD, Myf5, MyoG and MyHC relative mRNA levels. The levels of the myostatin-1 and myostatin-2 genes were downregulated, and the protein expression levels of p-Smad2, Smad2, Smad4, p-Akt, Akt, LARP6 and Smad3 were increased.

Conclusions: The appropriate levels of dietary protein promoted the growth of sub-adult grass carp and improved muscle hardness by promoting the growth of muscle fibers, improving collagen synthesis and depressing collagen degradation. In addition, the dietary protein requirements of sub-adult grass carp were 26.21% and 24.85% according to the quadratic regression analysis of growth performance (SGR) and the muscle hardness (collagen content), respectively.

Keywords: Collagen degradation; Collagen synthesis; Grass carp; Muscle fibers; Muscle hardness; Protein.

Fig. 1

The morphology Histological analysis in grass carp muscle. A Transection stained by H&E

Fig. 2

Heat map of related gene expression in muscle of grass carp. * Collagen metabolism-related signal molecules; # Factors related to muscle fibre growth. Red indicates high expression and n = 6 for each group

Fig. 3

Western blot analysis of relative protein expression in the muscle of grass carp. A Collagen transcription related factors; B Related factors regulating collagen mRNA translation; C Collagen degradation related factors. Values are means ± SD and n = 6 for each group. Different letters are significantly different (P < 0.05)