A new insight on copper: Promotion of collagen synthesis and myofiber growth and development in juvenile grass carp (Ctenopharyngodon idella)

Abstract

Copper (Cu) is a trace element, essential for fish growth. In the current study, in addition to growth performance, we first explored the effects of Cu on collagen synthesis and myofiber growth and development in juvenile grass carp (Ctenopharyngodon idella). A total of 1080 fish (11.16 ± 0.01 g) were randomly divided into 6 treatments (3 replicates per treatment) to receive five doses of organic Cu, which were Cu citrate (CuCit) at 0.99 (basal diet), 2.19, 4.06, 6.15, and 8.07 mg/kg, and one dose of inorganic Cu (CuSO₄·5H₂O at 3.15 mg/kg), for 9 weeks. The results showed appropriate Cu level (4.06 mg/kg) enhanced growth performance, improved nutritional Cu status, and downregulated Cu-transporting ATPase 1 mRNA levels in the hepatopancreas, intestine, and muscle of juvenile grass carp. Meanwhile, collagen content in fish muscle was increased after Cu intake, which was probably due to the following pathways: (1) activating CTGF/TGF-β1/Smads signaling pathway to regulate collagen transcription; (2) upregulating of La ribonucleoprotein domain family 6 (LARP6) mRNA levels to regulate translation initiation; (3) increasing proline hydroxylase, lysine hydroxylase, and lysine oxidase activities to regulate posttranslational modifications. In addition, optimal Cu group increased myofiber diameters and the frequency of myofibers with diameter >50 μm, which might be associated with upregulation of cyclin B, cyclin D, cyclin E, proliferating cell nuclear antigen, myogenic determining factor (MyoD), myogenic factor 5, myogenin (MyoG), myogenic regulatory factor 4 and myosin heavy chain (MyHC) and downregulation of myostatin mRNA levels, increasing protein levels of MyoD, MyoG and MyHC in fish muscle. Finally, based on percentage weight gain (PWG), serum ceruloplasmin (Cp) activity and collagen content in fish muscle, Cu requirements were determined as 4.74, 4.37 and 4.62 mg/kg diet (CuCit as Cu source) of juvenile grass carp, respectively. Based on PWG and Cp activity, compared to CuSO₄·5H₂O, the efficacy of CuCit were 131.80% and 115.38%, respectively. Our findings provide new insights into Cu supplementation to promote muscle growth in fish, and help improve the overall productivity of aquaculture.

Keywords: Collagen synthesis; Copper; Grass carp (Ctenopharyngodon idella); Myofiber.

Fig. 1

Effects of different Cu levels on Cu content in hepatopancreas, serum and muscle (A) and the Cu transporter ATP7A mRNA expression (B) in hepatopancreas, intestine and muscle of juvenile grass carp. Mean values with different superscript letters indicate significant difference (one-way ANOVA and Duncan's multiple-range tests, P < 0.05). The asterisk (∗) indicates a significant difference between 0.99 mg/kg CuCit and CuSO₄·5H₂O groups (P < 0.05); the number sign (^#) indicates a significant difference between 4.06 mg/kg CuCit and CuSO₄·5H₂O groups (P < 0.05). Values are means ± SD, n = 3 (for 3 replicate groups, 2 fish per replicate). ATP7A = Cu-transporting ATPase 1.

Fig. 2

Effects of different Cu levels on collagen deposition in muscle of juvenile grass carp. (A) Sirius red staining of muscle tissue in fish (magnification 200× ; scale bar = 100 μm). Collagen was stained by red color (n = 3). (B) The proportion of collagen fibers according to statistical analysis of the Sirius red staining (n = 3). (C) Collagen content calculated from hydroxyproline content. (D) Proline hydroxylase (PHD; U/g tissue). (E) Lysine hydroxylase (LH; U/g tissue). (F) Lysine oxidase (LOX; IU/g tissue). (G) Relative mRNA expression. Col1α1 = type I collagen α1; Col1α2 = type I collagen α2; LARP6 = La Ribonucleoprotein 6; Sp1 = specificity protein 1; CTGF = connective tissue growth factor; TGF-β1 = transforming growth factor-β1. (H–I) Western blot analysis of collagen synthesis in the muscle of juvenile grass carp fed diets containing different levels of Cu for 9 weeks. The fold changes are based on the mRNA levels of different genes. Mean values with different superscript letters indicate significant difference (one-way ANOVA and Duncan's multiple-range tests, P < 0.05. The asterisk (∗) indicates a significant difference between 0.99 mg/kg CuCit and CuSO₄·5H₂O groups (P < 0.05); the number sign (^#) indicates a significant difference between 4.06 mg/kg CuCit and CuSO₄·5H₂O groups (P < 0.05). Values are means ± SD, n = 3 (for 3 replicate groups, 2 fish per replicate).

Fig. 3

Effects of different Cu levels on the growth and development of myofiber in juvenile grass carp muscle. (A) Transversal section micro-structure (magnification 200× , scale bar = 100 μm) of muscle in juvenile grass carp supplemented with different Cu levels for 9 weeks visualized using H&E staining. (B) Myofiber diameters (n = 3). (C) Frequency of distribution (%) of the diameters of myofiber (n = 3). (D) Relative mRNA expressions and (E) Western blot analysis of myogenic regulatory factors in the muscle of juvenile grass carp fed diets containing different Cu levels for 9 weeks. The fold changes are based on the mRNA expression levels of different genes. Mean values with different superscript letters indicate significant difference (one-way ANOVA and Duncan's multiple-range tests, P < 0.05). The asterisk (∗) indicates a significant difference between 0.99 mg/kg CuCit and CuSO₄·5H₂O groups (P < 0.05); the number sign (^#) indicates a significant difference between 4.06 mg/kg CuCit and CuSO₄·5H₂O groups (P < 0.05). Values are means ± SD, n = 3 (for 3 replicate groups, 2 fish per replicate).

Fig. 4

Quadratic regression analysis of (A) percentage weight gain (PWG), (B) ceruloplasmin activity, and (C) collagen content in juvenile grass carp fed diets with various Cu levels for 9 weeks.