Implications of vitamin D for flesh quality of grass carp (Ctenopharyngodon idella): antioxidant ability, nutritional value, sensory quality, and myofiber characteristics

Abstract

Background: Muscle represents a unique and complex system with many components and comprises the major edible part of animals. Vitamin D is a critical nutrient for animals and is known to enhance calcium absorption and immune response. In recent years, dietary vitamin D supplementation in livestock has received increased attention due to biological responses including improving shear force in mammalian meat. However, the vitamin D acquisition and myofiber development processes in fish differ from those in mammals, and the effect of vitamin D on fish flesh quality is poorly understood. Here, the influence of dietary vitamin D on fillet quality, antioxidant ability, and myofiber development was examined in grass carp (Ctenopharyngodon idella).

Methods: A total of 540 healthy grass carp, with an initial average body weight of 257.24 ± 0.63 g, were allotted in 6 experimental groups with 3 replicates each, and respectively fed corresponding diets with 15.2, 364.3, 782.5, 1,167.9, 1,573.8, and 1,980.1 IU/kg vitamin D for 70 d.

Results: Supplementation with 1,167.9 IU/kg vitamin D significantly improved nutritional value and sensory quality of fillets, enhancing crude protein, free amino acid, lipid, and collagen contents; maintaining an ideal pH; and reducing lactate content, shear force, and cooking loss relative to respective values in the control (15.2 IU/kg) group. Average myofiber diameter and the frequency of myofibers > 50 μm in diameter increased under supplementation with 782.5-1,167.9 IU/kg vitamin D. Levels of oxidative damage biomarkers decreased, and the expression of antioxidant enzymes and nuclear factor erythroid 2-related factor 2 signaling molecules was upregulated in the 1,167.9 IU/kg vitamin D treatment compared to respective values in the control group. Furthermore, vitamin D supplementation activated cell differentiation by enhancing the expression of myogenic regulatory factors and myocyte enhancer factors compared to that in the control group. In addition, supplementation with 1,167.9 IU/kg vitamin D improved protein deposition associated with protein synthesis molecule (target of rapamycin) signaling and vitamin D receptor paralogs, along with inhibition of protein degradation (forkhead box protein 1) signaling.

Conclusions: Overall, the results demonstrated that vitamin D strengthened antioxidant ability and myofiber development, thereby enhancing nutritional value and sensory quality of fish flesh. These findings suggest that dietary vitamin D supplementation is conducive to the production of nutrient-rich, high quality aquaculture products.

Keywords: Antioxidant; Grass carp; Myofiber development; Nutritional value; Sensory quality; Vitamin D.

Fig. 1

Proximate composition and physicochemical properties of the muscle from grass carp (Ctenopharyngodon idella) after a 70-day growth trial. A Moisture content; B Crude protein content; C Crude lipid content; D Ash content; E pH; F Lactate content; G Shear force; H Collagen content; I Hydroxyproline level; J Cooking loss; K Cathepsin B activity; L Cathepsin L activity in fillets. Results are shown as means ± SD (n = 6). Different letters above bars indicate significant differences (P < 0.05)

Fig. 2

Effects of vitamin D on the activities of antioxidant enzymes and glutathione (GSH) in the muscle of grass carp (n = 6). A Protein carbonyl (PC); B Malondialdehyde (MDA); C Reactive oxygen species (ROS); D Total antioxidant capacity (T-AOC); E Superoxide dismutase (SOD); F CuZnSOD; G MnSOD; H Catalase (CAT); I Glutathione peroxidase (GPx); J Glutathione reductase (GR); K Glutathione S-transferase (GST); L Antihydroxyl radical (AHR); M Anti-superoxide anion (ASA); N GSH. Values are shown as means ± SD. Results with different superscripts are significantly different (P < 0.05)

Fig. 3

Effects of vitamin D on gene and protein levels of antioxidant parameters in the muscle of grass carp (n = 6). A Relative mRNA levels of antioxidant enzymes; B Relative mRNA levels of NF-E2-related factor 2 (Nrf2) signaling; C Protein level of Nrf2. Values are shown as means ± SD. Results with different superscripts are significantly different (P < 0.05)

Fig. 4

Histological observation of myofibers in grass carp fed different levels of vitamin D. A Hematoxylin-eosin staining results in cross-sections of fish muscle (200×; n = 6); B Myofiber diameter; C Frequency of distribution (%) of myofibers in different diameter classes; D Transmission electron microscope image; E Sarcomere length of myofibers in fish supplemented with 15.2 (control) and 1,167.9 IU/kg vitamin D (n = 3). M line, Z disk, and sarcomere are shown to represent myofiber structure. Values are shown as means ± SD. Different letters above bars indicate significant differences (P < 0.05)

Fig. 5

Effects of vitamin D on gene (A) and protein levels (B and C) of myogenic regulatory factors and myocyte enhancement factors in the muscle of grass carp (n = 6). Values are shown as means ± SD. Results with different superscripts are significantly different (P < 0.05). MyoD, myogenic differentiation antigen; MRF4, myogenic regulatory factor 4; Myf5, myogenic factor 5; MyoG, myogenin; MEF, myocyte enhancer factor

Fig. 6

Effects of vitamin D on the transcription (A) and protein levels (B and C) of protein synthesis signaling molecules in the muscle of grass carp (n = 6). Values are shown as means ± SD. Results with different superscripts are significantly different (P < 0.05). TOR, target of rapamycin; S6K1, ribosomal protein S6 kinase; 4E-BP1, eIF4E-binding protein 1

Fig. 7

Effects of vitamin D on protein degradation in the muscle of grass carp. A SDS-PAGE results of muscle protein extract in different concentration separation gel. M, marker; numbers 1–6 represent different vitamin D levels from 15.2 to 1,980.1 IU/kg (n = 3). B and C Effects of dietary vitamin D on the transcription (B) and protein levels (C) of autophagy-lysosome pathway, ubiquitin-proteasome pathway, and FOXO molecules (n = 6). D UB protein level. E Immunofluorescence results of FoxOX1 in the 15.2 and 1,167.9 IU/kg groups (n = 3). The white arrow shows nuclear co-localization. Values are shown as means ± SD. Results with different superscripts are significantly different (P < 0.05). ATG, autophagy-related gene; LC3, light chain 3; MuRF1, muscle-specific ring finger; MAFbx, muscle atrophy F-box; UB, ubiquitin; FoxO, forkhead box O

Fig. 8

Effects of vitamin D on the transcription levels of AKT, TSC2, and VDR (A), and on protein levels of VDR (B) in the muscles of grass carp (n = 6). C Immunofluorescence results for VDR in 15.2 and 1,167.9 IU/kg treatment groups. The white arrow shows nuclear co-localization (n = 3). Values are shown as means ± SD. Results with different superscripts are significantly different (P < 0.05). AKT, protein kinase B; TSC2, tuberous sclerosis 2; VDR, vitamin D receptor

Fig. 9

Quadratic regression analysis of crude protein (A) and cooking loss (B) in the fillets of grass carp (Ctenopharyngodon idella) after receiving different levels of dietary vitamin D