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

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.


a b s t r a c t
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 (CuSO 4 $5H 2 O 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-b1/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 mm, 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 CuSO 4 $5H 2 O, the efficacy

Introduction
Copper (Cu), as a trace mineral element, is essential for several biological processes, including hemoglobin synthesis, bone formation, and myelin maintenance in the nervous system, and it also acts as a substance of key enzymes such as cytochrome oxidase and CueZn superoxide dismutase enzyme (Gaetke et al., 2014).Cu is also crucial for fish growth (Lall, 2003).It has been shown that suitable Cu levels improve the growth performance of on-growing grass carp (Ctenopharyngodon idella) (Tang et al., 2013) and Indian juvenile carp (Labeo rohita) (Musharraf and Khan, 2022).Animal growth is primarily determined by skeletal muscle development, which is inseparable from myofiber growth and development, as well as extracellular matrix (ECM) deposition (Karalaki et al., 2009).Collagen is the most plentiful ECM element in skeletal muscle tissue (Mukund et al., 2017) and is essential for muscle development and structural stability (Csapo et al., 2020).A previous study showed that Cu deficiency led to impairment of collagen crosslinking and maturation in rat hearts (Werman and David, 1996).In addition, Cu had a certain effect on the proliferation and differentiation of mouse primary myoblasts (Vest et al., 2018).It was indicated Cu influenced collagen synthesis and myofiber growth and development.Nevertheless, to date, the effects of Cu on collagen synthesis and myofiber growth and development have not been reported in fish.Therefore, systematic, and in-depth research is essential.
Collagen synthesis in vertebrates generally involves transcription, translation, and posttranslational modifications (Yu et al., 2019).Collagen transcription and translation initiation are regulated by the transforming growth factor-b1 (TGF-b1)/Smads signaling pathway (Ma et al., 2020), and La ribonucleoprotein domain family 6 (LARP6) (Stefanovic et al., 2022), respectively.In addition, collagen posttranslational modifications are partially regulated by proline hydroxylase (PHD) (Yang et al., 2022), lysine hydroxylase (LH) (Wan et al., 2020) and lysine oxidase (LOX) (Trackman et al., 2015).Studies on LOX have been reported.It showed that Cu deficiency downregulated LOX mRNA levels in goat hearts, leading to impaired collagen cross-linking (Mandour et al., 2021).However, the regulation of collagen transcriptional, translational, and posttranslational modifications (except for LOX) by Cu have not been studied in animals.Cu is crucial for the transfer and metabolism of iron (Sharp, 2004).A study in our laboratory showed that suitable iron upregulated TGF-b1 mRNA levels in grass carp skin (Guo et al., 2017).Meanwhile, suitable iron elevated PHD activity in mouse hepatoma cells (Martin et al., 2005).In addition, Cu upregulated insulin-like growth factor-1 (IGF-1) mRNA levels in human cardiomyocytes (Jiang et al., 2007), while IGF-1 could upregulate LARP6 mRNA levels in mouse smooth muscle (Blackstock et al., 2014).These findings implied that Cu might have an impact on collagen synthesis in animal muscle, which needs to be explored.
As an important component in the ECM, collagen enhances adhesion kinase activity and thus stimulates myofiber growth and development (Liu et al., 2020).Unlike terrestrial animals, the myofiber growth and development of fish mainly depends on proliferation and hypertrophy (Valente et al., 2013).Myofiber proliferation and hypertrophy are regulated by cell cycle proteins (such as cyclin B and cyclin D) (Mastroyiannopoulos et al., 2012) and myogenic determinants (such as myogenic determining factor (MyoD), myogenin (MyoG), and myosin heavy chain (MyHC)) (Zammit, 2017), respectively.Nevertheless, the effects and regulatory mechanisms of Cu on myofiber proliferation and hypertrophy have not been reported in animals.A previous study reported that Cu upregulated antioxidant-1 (ATOX-1) mRNA levels in finishing pig liver (Wen et al., 2022), and ATOX-1 upregulated cyclin B1 and cyclin D1 mRNA levels in mouse embryonic fibroblasts (Itoh et al., 2008).In addition, Cu downregulated Cu-transporting ATPase 1 (ATP7A) mRNA levels in finishing pig jejunal mucosa (Wen et al., 2022), and low levels of ATP7A upregulated MyoD, MyoG and MyHC mRNA levels in mouse myogenic cells (Abdelsaid et al., 2022;Gabay et al., 2020).Therefore, we speculated that Cu might promote skeletal muscle growth related to cell proliferation regulators and myogenic differentiation factors in fish, which needs to be studied.
In general, dietary Cu sources include inorganic (such as CuSO 4 $5H 2 O) and organic Cu in fish.Cu citrate (CuCit), as a new organic Cu source, has the advantages of good safety, low addition and emission, easy absorption, and high utilization rate in the animal body.CuCit can effectively provide Cu elements and protect the ecological environment (Yan et al., 2015).Grass carp is native to China and has been oriented to many countries, it is one of the most critical species for freshwater aquaculture (Wu et al., 2012).At present, the Cu requirements of grass carp have only been reported in our laboratory, and CuSO 4 $5H 2 O was used as the Cu source (Tang et al., 2013).However, different Cu sources have different Cu requirements.The Cu requirements of juvenile grouper (Epinephelus malabaricus) supplemented with CuSO 4 $5H 2 O (5.36 mg/kg) (Lin et al., 2008) was higher than that of Cu peptide (organic Cu, 2.19 mg/kg) based on weight gain (Lin et al., 2010).In addition, there are differences in the bioavailability of different Cu sources.Studies have shown that 125 mg/kg Cu (CuCit) and 250 mg/kg Cu (CuSO 4 ) were equally effective in weaned pig (Armstrong et al., 2004) and broiler chicken (Pesti and Bakalli, 1996) growth.Therefore, the bioavailability of CuCit might be higher than that of CuSO 4 .However, no studies have evaluated dietary Cu requirements (CuCit as Cu source) and the efficacy of CuCit in comparison to CuSO 4 $5H 2 O of grass carp, which is crucial to investigate.
In summary, we first investigated the influences of Cu on collagen synthesis (except for LOX) and related signaling pathways, as well as on myofiber proliferation and hypertrophy in juvenile grass carp.The study might offer a rudimentary theoretical foundation for the regulation and mechanism of Cu on fish skeletal muscle growth.Cu requirements (CuCit as Cu source), and CuCit bioavailability relative to CuSO 4 $5H 2 O of juvenile grass carp were also determined to provide a reference for commercial formulation feed for fish.

Animal ethics statement
All the procedures in this study were authorized by the Institutional Animal Care Advisory Committee of Sichuan Agricultural University (No. MR-2020314085).

Experimental design and diets
Table 1 displays the ingredients in the basal diet.Protein sources were mainly composed of casein, gelatin, and wheat gluten, while lipid sources were mainly consisted of fish oil and soybean oil.The basal diet was formulated to contain approximately crude protein at 324.34 g/kg diet and crude lipid at 47.87 g/kg diet, which were measured by the Kjeldahl method and the Soxhlet exhaustive extraction technique respectively using the standard methodology (AOAC, 1995).Calcium and total phosphorus were 5.27 and 15.48 g/ kg diet, respectively, and were determined according to the national standard Determination of Calcium in Feed (GB/T 6436-2018) and Determination of total phosphorus in feed (GB/T 6437-2018), respectively.The crude fiber and gross energy were measured using a fiber analyzer (A2000I, ANKOM Technology, New York, USA) and oxygen bomb calorimeter (6400, Parr Instrument Company, Moline, USA), respectively, according to the method described by Zhang et al. (2016).The n-3 and n-6 PUFA were calculated referring to Zeng et al. (2016).The available phosphorus was calculated by NRC (National Research Council, 2011).Two Cu sources (Sichuan Animal Feed Co., Ltd.) were used: CuCit (Cu purity 34.5%) and CuSO 4 $5H 2 O (Cu purity 24.5%).The experiment used the following treatment structure: 1 (Cu-free control) þ 4 (CuCit) þ1 (CuSO 4 $5H 2 O).The Cu-free diet served as the control group, in which Cu content was at 0.96 mg/kg diet (calculated by measuring Cu contents in the ingredients with atomic absorption spectrometry).Cu was added to the test diets to provide graded Cu concentrations of 0.96 (un-supplemented control, Cu-deficient), 2.00, 4.00, 6.00, 8.00 and 3.00 mg/kg diet.The final Cu contents in the six treatments were determined to be 0.99 (un-supplemented control, Cu-deficient) and 2. 19, 4.06, 6.15, 8.07 and 3.15 mg/kg diet.The diets were made employing the method described by Mai et al. (2009).In short, the ingredients were ground through a 300-mm sieve into a fine powder.The oil and water were added to the premixed dry ingredients, mixed well, squeezed through an extruder with a mold and air-dried at room temperature.According to Wang et al. (2016), the diets were broken up, sifted into pellets, and stored at À20 C.

Feeding management
A local farm provided juvenile grass carp (Chengdu, China).Experimental fish were reared in outdoor freshwater ponds and fed five times per day (08:00, 11:00, 13:00, 15:00 and 19:00).Prior to the trial, they were fed a commercial diet at a rate of 4% of initial body weight for 4 weeks to acclimatize to the experimental environment and were then fed a basal diet (Cu-free) for 2 weeks to decrease Cu content according to Tang et al. (2013).Later, a total of 1080 fish were randomly divided into 18 net cages (150 cm Â 150 cm Â 150 cm, 60 fish per cage) with an initial mean weight of 11.16 g/fish, and divided into six treatments, each with three replicates.As described by Wu et al. (2017), we fitted a disc with a diameter of 80 cm to the bottom of each cage and wrapped the disc with 1-mm gauze to collect the remaining feed.During a 9-week experiment, the corresponding experimental feed was fed until saturated.After feeding for 30 min, the uneaten feed was siphoned out, dried, and weighed.Feed intake was calculated on a dry matter basis according to the method described by Cai et al. (2005).For culture water impurity removal and ammonia concentration reduction, water was pumped through a sand filter at a rate of 1 L/min in each cage according to Wu et al. (2017) method.Throughout the trial period, microporous aeration was used, and water was changed daily.We used a professional multiparameter instrument (YSI Incorporated, Yel-low Springs, OH, USA) to measure water quality every day.Water temperature and pH value were determined at 27.8 ± 3 C and 7.5 ± 0.4, respectively, and dissolved oxygen was !6.0 mg/L.Cu concentration in the culture water was determined to be 5 mg/L in accordance with the Standard test methods for drinking water -metal indicators (GB/T 5750.6-2006).

Sample collection and analysis
After the feeding trial, all fish were starved for 24 h and given a benzocaine bath (50.0 mg/L) for anesthesia.At the start of sampling, the body weight, length, width, and height were measured prior to blood collection.Then, blood was collected from the tail vein of fish, centrifuged, and the supernatant was removed and stored at À80 C according to Wang et al. (2015).Fish were killed to collect samples in the hepatopancreas, intestine and muscle.Tissue samples for body composition, Cu contents, enzyme activities, mRNA and protein level analysis were frozen with liquid N 2 and then kept at À80 C. Muscle samples for sectioning analysis were stored in 4% para-formaldehyde.
The approximate composition of whole fish and muscle were determined using the standard methodology (AOAC, 1995).The cooking loss, pH value and shear force of muscle were determined by applying Wang et al. (2015) method.Hydroxyproline (HYP) content was analyzed with kits obtained from China's Nanjing Jiancheng Bioengineering Research Institute.The collagen content was calculated by multiplying the HYP content by 8 based on the approach of Sun et al. (2017).

Biochemical analysis
Cu contents in hepatopancreas, serum and muscle were determined by atomic absorption spectrometry (CONTAA700, Jena Analytical Instruments AG, Germany) according to National Standard for Safety Determination of Cu in Food (GB 5009.13-2017).Serum ceruloplasmin (Cp) activity was analyzed with kits obtained from China's Nanjing Jiancheng Bioengineering Research Institute, which was measured using o-dianisidine dihydrochloride as a substrate.Muscle tissue was weighed 100 mg, homogenized in 0.9% sterile saline (1:10, wt/vol), subsequently incubated on ice for 30 min and centrifuged at 6000 Â g for 20 min at 4 C to collect the supernatant to obtain a muscle tissue homogenate, which was used to measure muscle enzyme activities (Wang et al., 2015).Enzyme activities such as PHD, LH and LOX were measured using ELISA kits (Shanghai Changjin Biotechnology Co., Ltd, Shanghai, China).

Histological analysis
Muscle samples were routinely paraffin embedded and sectioned after being treated with 4% paraformaldehyde.
Hematoxylin-eosin (H&E) staining was used to stain 4 mm paraffin sections.ImageJ software was used to analyze the myofiber mean diameters of three images (for each picture, 300 myofibers were chosen at random).The frequency of various myofiber sizes (<20, 20 to 50, and >50 mm) was determined employing the method of Valente et al. (2016).
Adopting the method of Lin et al. ( 2022), using Sirius red staining, the collagen fiber area was examined in grass carp muscle.
Briefly, muscle paraffin slices (4 mm) were dewaxed, rehydrated, and then stained with Sirius red solution sets (1 g/L Sirius red F3B in picric acid for 90 min, and 0.01 M HCl for 10 min, respectively).The collagen region was evaluated using ImageJ software (3 complete images for each group; red; %).All sections were observed using an upright light microscope (Japan, Nikon).

Real-time PCR analysis
The real-time PCR procedure was performed using the method of Ma et al. (2020) in our laboratory.In brief, an RNA iso Plus kit (Dalian Takara, China) was used to isolate total RNA from hepatopancreas, intestinal and muscle tissues.Next, total RNA was treated with DNAse I. RNA quality and quantity were determined by electrophoresis on 1% agarose gels and Nanodrop 2000 (Thermo Fisher Scientific, Wilmington, DE, USA), respectively.Additionally, the PrimeScript RT kit was used to reverse transcribe RNA to cDNA.Primer 5.0 software was used to build the primers using the Sus-Scrofa sequences in the NCBI database and the primers were synthesized by Bioengineering (Shanghai) Co., Ltd.The primers are listed in Table S1.Beta-actin was used as an endogenous control based on selection.The gene expression results were measured by the 2 ÀDDCt method.

Western blotting
The previous method described by Dong et al. (2018) was adopted to analyze Western blotting.Briefly, RIPA lysis buffer (Chinese Beyotime) was used to extract total muscle protein, and then protein concentrations were measured using a BCA assay kit (Beyotime Biotechnology Inc., China).Then, SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was applied, and the target proteins (40 mg per sample) were separated and transferred to polyvinylidene-fluoride (PVDF) membranes.The PVDF membranes were blocked for 2 h at room temperature, followed by overnight incubation with primary antibodies (TGF-b1, Col1a1, Col1a2, total-Smad2, p-Smad2 Ser467, Smad3, Smad4, MyoD, MyoG, MyHC and b-actin) at 4 C.These antibodies were purchased from Ebtek Biotechnology Co., Ltd.The following day, the blots were washed 3 times and then incubated with the enzyme-labeled secondary antibody (goat anti-rabbit, Santa Cruz Biotechnology, Santa Cruz, CA, USA) in Tris Buffered Saline with Tween 20 for 2 h.Finally, Enhanced chemiluminescence kit (Beyotime Biotechnology, Inc., China) was used for imaging, and Image Lab 6.1 was used to view the immunological complex.

Calculating and statistical analyses
Table 2 summarizes the formulas of the growth performance indices.The mean ± standard deviation (SD) was used to express the results.SPSS software was used to conduct statistical analysis.Using one-way ANOVA and Duncan's multiple range test, the Cudeficient and CuCit groups were compared.Then, an independent sample t-test was used for Cu-deficient group and CuSO 4 $5H 2 O group, and an independent sample t-test was used for CuCit optimal group and CuSO 4 $5H 2 O group.Significance was considered with P < 0.05.We conducted unary linear regression, quadratic regression, and triple regression for comparison to predict a more accurate response to the dietary intake based on the results of R 2 and P-value using a quadratic regression model to calculate the dietary Cu requirements of the fish.

Growth performance, Cu status and chemical composition in juvenile grass carp
Table 3 lists the growth parameters.There was no significant difference in the mean initial body weight of grass carp among all groups (P > 0.05).With increasing Cu levels up to 4.06 mg/kg, the final body weight (FBW), percentage weight gain (PWG), specific growth rate (SGR), feed intake (FI), and feed efficiency (FE), total length, body length, width and height were significantly higher than those of the Cu-deficient group (P < 0.05).The total length, width, and height of the body at the 4.06 mg/kg CuCit level were significantly higher than those at the CuSO 4 $5H 2 O level (P < 0.05).In addition, condition factor (CF) increased significantly (P < 0.05) as the Cu levels increased to 2.19 mg/kg compared to the Cu deficient group.Nevertheless, Cu did not affect the survival rate of juvenile grass carp (P > 0.05).With the enhancement of Cu levels, serum Cp activity rose sharply (P < 0.05) and peaked at 4.06 mg/kg Cu level.At the same time, Cp activity was notably higher at    juvenile grass carp hepatopancreas, intestine, and muscle ATP7A mRNA levels, which were markedly reduced with dietary Cu levels reaching 4.06 mg/kg (P < 0.05).At the same time, the ATP7A mRNA level in the intestine was lower in the 4.06 mg/kg CuCit group than in the CuSO 4 $5H 2 O group (P < 0.05).
The effects of Cu on the whole-body and muscle nutritional value of juvenile grass carp are shown in Table 4.When fish were fed the diet with 4.06 mg/kg Cu level, the protein and lipid contents in the whole body reached the highest value and dropped as Cu levels increased (P < 0.05).With the enhancement of Cu levels, both protein and lipid production values (PPV, LPV) had a significant effect, which significantly increased before 4.06 mg/kg Cu level (P < 0.05) and then declined dramatically at higher levels (P < 0.05).In contrast, whole-body moisture reached its lowest value at 4.06 mg/kg Cu level.Moreover, both crude protein and lipid contents in fish muscle increased significantly with increasing Cu levels (P < 0.05), and the maximum value were 21.64 and 3.42 at 4.06 mg/ kg Cu level, respectively.Juvenile grass carp muscle moisture was unaffected by Cu.
The cooking loss decreased significantly with Cu levels rising to 4.06 mg/kg (P < 0.05), which at 4.06 mg/kg CuCit level was lower than CuSO 4 $5H 2 O group.The shear force, pH and HYP content reached a maximum at 4.06 mg/kg CuCit and their values at 4.06 mg/kg were higher than those of the CuSO 4 $5H 2 O group (P < 0.05).

Collagen-associated indicators in juvenile grass carp muscle
Fig. 2 depicts that Cu affects the collagen-related indicators in juvenile grass carp.Sirius red staining was used to analyze the effect of Cu levels on collagen deposition in juvenile grass carp muscle (Fig. 2AeB).The results demonstrated that the collagen fiber area (red) increased with increasing Cu levels when compared to the Cu-deficient group, reaching a maximum at 4.06 mg/kg (P < 0.05).Furthermore, the 4.06 mg/kg CuCit group was notably higher than the CuSO 4 $5H 2 O group (P < 0.05).
As shown in Fig. 2CeF, compared with the Cu-deficient group, the collagen content and PHD, LH and LOX activities in added Cu treatments were significantly increased (P < 0.05) and maximized at 4.06 mg/kg level.In addition, these indices in the 4.06 mg/kg CuCit level were higher than those in the CuSO 4 $5H 2 O group.

Myofiber growth and development-associated indicators in juvenile grass carp muscle
Fig. 3AeC illustrates that Cu affects the myofiber characteristics of juvenile grass carp.The myofiber diameters of the 2.19, 4.06, 6.15 and 8.07 mg/kg Cu groups were considerably higher than those of the Cu-deficient group, and the frequency of myofibers >50 mm diameter showed the same trend as it (P < 0.05).Compared to the Cu-deficient group, the frequency of <20 and 20e50 mm diameter were markedly reduced in the other groups (P < 0.05).
Fig. 3DeE shows the effects of Cu on mRNA and protein levels associated with proliferation and differentiation of myogenic cells in juvenile grass carp muscle.Compared to the Cu-deficient group, the mRNA levels of cyclin B, cyclin D, cyclin E, proliferating cell nuclear antigen (PCNA), MyoG, MyoD, Myf5, MRF4 and MyHC at 4.06 mg/kg Cu level were significantly increased (P < 0.05) and then progressively declined.In contrast, as Cu levels increased, significantly lowered MSTN mRNA levels were observed (P < 0.05) and reached the lowest value at 4.06 mg/kg Cu level, and then steadily rose.Additionally, the protein levels of MyoG, MyoD, and MyHC increased significantly with Cu levels reaching 4.06 mg/kg diet (P < 0.05).

Cu improved growth performance and Cu nutritional status of fish
This study showed that growth performance of juvenile grass carp was significantly influenced by appropriate dietary Cu.According to our findings, the PWG, FI, and FE of juvenile grass carp were increased by appropriate dietary Cu, proving that appropriate Cu level (4.06 mg/kg) could enhance the growth performance of juvenile grass carp, which was consistent with on-growing grass carp (Tang et al., 2013) and Indian juvenile carp (Musharraf and Khan, 2022).
Nutrient status in the fish influences their growth (Wei et al., 2018).Serum and tissue Cu levels sensitively reflect Cu nutritional status (Shao et al., 2010).Serum Cp, a member of the poly Cu oxidase family, is an enzyme with oxidase activity (Hellman and Gitlin, 2002).Serum Cp is also a good indicator of Cu nutritional status (Merle et al., 2009).Our findings demonstrated that supplemental Cu raised the hepatopancreas, serum, and muscle Cu concentrations, as well as serum Cp activity of juvenile grass carp, demonstrating that dietary Cu could improve Cu nutritional status, which might be related to ATP7A.ATP7A belongs to the Cu-ATPase P 18 family and plays a vital role in the regulation of both cellular and systemic Cu homeostasis, which can transfer Cu from intracellular to extracellular (Lutsenko et al., 2007).Our findings demonstrated that appropriate Cu level decreased ATP7A mRNA abundance in the hepatopancreas, intestine, and muscle.The same results were obtained in finishing pigs (Wen et al., 2022).The outcomes listed above showed that cellular Cu export demand was reduced at optimal Cu concentrations, regulating cellular Cu flux, and thus maintaining Cu homeostasis.
Fish growth depends on nutrient deposition (Hua et al., 2007).Our results showed that suitable Cu level could increase the protein and lipid contents and PRV as well as LRV of the whole body in juvenile grass carp, which supported earlier findings in red sea bream (El Basuini et al., 2016) and juvenile sturgeon (Wang et al., 2016).Muscle tissue is the main edible part of the fish body (Wei et al., 2016).The nutritional contents (such as proteins and lipids) and physicochemical properties of muscle (such as shear force, pH and cooking loss) are important for assessing muscle nutritional value and muscle quality (Mir et al., 2017).In this research, we found that appropriate Cu boosted protein and lipid contents, improved shear force and pH, and reduced cooking loss in juvenile grass carp muscle.In summary, the physicochemical characteristics and nutritional value of juvenile grass carp were improved by suitable Cu level.
As mentioned before, animal growth is closely related to muscle growth and development, and collagen synthesis is closely linked to muscle growth, whereas our research demonstrated that Cu could enter the muscle, so we subsequently explored the effects of Cu on collagen synthesis and myofiber growth and development in juvenile grass carp.

Cu increased collagen content related to the regulation of collagen synthesis in fish muscle
Collagen makes up 3% to 10% of the protein in fish muscle (Delbarre et al., 2006).By multiplying the HYP content by 8, the collagen content can be estimated (AOAC, 2006).Meanwhile, Sirius red staining is one of the staining methods for collagen fibers, which are colored red (Hyllested et al., 2002).Our results demonstrated that appropriate Cu level increased HYP contents and collagen fibers by Sirius red staining in juvenile grass carp muscle, indicating that Cu increased the collagen content of fish muscle.Collagen content is largely influenced by collagen synthesis.In fact, collagen synthesis is closely related to transcription, translation initiation and posttranslational modifications (Gelse et al., 2003).Therefore, determining the effects of Cu on collagen transcription, translation initiation, and posttranslational modifications was the next step of our research.
Most of type I collagen was found in grass carp muscle.Two a1 chains (Col1a1) and one a2 chain (Col1a2) make up type I collagen (Sun et al., 2018).Our findings revealed that Col1a1 and Col1a2 mRNA and protein levels were elevated by suitable Cu level, and the TGF-b1/Smads signaling pathway might be responsible for this result.The TGF-b1/Smads signaling pathway regulates type I collagen transcription (Yu et al., 2019).TGF-b1 binds to its receptor, recruits, and phosphorylates Smad2 and Smad3, forms a complex with Smad4, translocate into the nucleus as a transcription factor, and binds to target genes through other transcription factors such as Sp1 to initiate collagen synthesis, thus enhancing collagen deposition (Jinnin, 2010).Our results showed that appropriate Cu level upregulated the mRNA levels of TGF-b1, Smad2, Smad3, Smad4, and Sp1 and the protein levels of TGF-b1, p-Smad2, Smad3 and Smad4 in juvenile grass carp muscle.Further correlation analysis revealed that Col1a1 and Col1a2 protein levels were positively correlated with TGF-b1, p-Smad2, Smad3 and Smad4 protein levels (Table 5), supporting our hypothesis.TGF-b1 was elevated by Cu and might be associated with connective tissue growth factor (CTGF).CTGF acts as a cofactor of TGF-b1, which is synthesized by myoblasts and myofibers, thereby inducing of type I collagen synthesis (Kong et al., 2014).In this experiment, we found that appropriate Cu levels enhanced CTGF mRNA abundance in juvenile grass carp muscle, and correlation analysis revealed that TGF-b1 and CTGF mRNA levels were positively associated (Table 5).In conclusion, Cu-increased muscle collagen content might be connected to the CTGF/TGF-b1/Smads signaling pathway.
After transcription, Col1a1 and Col1a2 translation initiation is mediated by LARP6, a member of the RNA-binding protein superfamily, which specifically binds the 5 0 stem loop in Col1a1 and Col1a2 mRNAs (Stefanovic et al., 2022).Our results suggested that optimal dietary Cu level upregulated LARP6 mRNA levels in juvenile grass carp muscle.Further correlation analysis showed that HYP content and Col1a1, and Col1a2 protein levels were positively correlated with LARP6 mRNA levels (Table 5), indicating that improved collagen synthesis with optimal dietary Cu might be partly related to translation initiation.In addition to translation initiation, posttranslational modifications also play an important role in collagen synthesis.As mentioned previously, posttranslational modifications are partially influenced by PHD, LH and LOX.We found that optimal Cu enhanced PHD, LH and LOX activities in juvenile grass carp muscle, and further correlation analysis revealed a positive correlation between collagen content and PHD, LH and LOX activities (Table 5), demonstrating that improvement of collagen content by optimal dietary Cu might be related partially to its posttranslational modifications.
As mentioned earlier, collagen is the main ECM component.Lysine oxidase regulates ECM cross-linking in extracellular muscle connective tissue while promoting differentiation within myogenic progenitor cells, ultimately leading to myofiber growth and development (Gabay et al., 2020).Hence, we explored the mechanism whereby dietary Cu affects myofiber growth and development.
4.3.Cu promoted the myofiber growth of fish related to proliferation and differentiation of myoblasts Fish muscle growth and development are achieved by a combination of myofiber proliferation and hypertrophy (Valente et al., 2013).As fish body weight increases, myofiber diameters increase (Yu et al., 2017).The frequency of myofibers with diameter <20 and >50 mm represents a more active growth pattern of myofiber proliferation and hypertrophy (Silva et al., 2009).In the present study, we first revealed that appropriate dietary Cu decreased the frequency of myofibers with diameter <20 mm and increased the myofiber diameters and the frequency of myofibers with diameter >50 mm in juvenile grass carp, indicating that optimal dietary Cu promoted myofiber hypertrophy in fish.Fish myofiber growth and development is closely related to myoblast proliferation (Johnston et al., 2011).Proliferation is regulated by cell cycle proteins (such as cyclin B, cyclin D and cyclin E) (Mastroyiannopoulos et al., 2012) and PCNA (Shen et al., 2003).The results of the present study first showed that suitable Cu level upregulated cyclin B, cyclin D, cyclin E and PCNA mRNA levels in juvenile grass carp muscle, confirming that Cu-improved fish myofiber growth and development might be connected to promoting myoblast proliferation.
In addition to proliferation, myoblast differentiation also has a crucial function in myofiber growth and development.Myogenic regulatory factors (MyoD, Myf5, MyoG and MRF4) play a key role in animal myogenic cell differentiation (Sabourin and Rudnicki, 2000).MyHC is a marker protein of late differentiation of myogenic cells (Chu et al., 2010).In this experiment, we discovered that optimal Cu level increased MyoD, MyoG, Myf5, MRF4 and MyHC mRNA levels, as well as MyoD, MyoG and MyHC protein levels in juvenile grass carp muscle.Cu-upregulated myogenic regulators might be related to LOX.Lysyl oxidase can upregulate the mRNA levels of MyoD, Myf5, MyoG and MyHC in mouse myogenic progenitor cells (Gabay et al., 2020).Our findings revealed that appropriate Cu level increased LOX activity in juvenile grass carp muscle.The MyoD, Myf5 and MRF4 mRNA levels, as well as MyoG and MyHC protein levels were positively linked to LOX activity according to further correlation analysis (Table 5), confirming our assumption.In addition, myofiber growth was negatively regulated by MSTN, which inhibited C2C12 myogenic cell proliferation and differentiation (Langley et al., 2002).Our research revealed that suitable Cu level decreased MSTN mRNA levels in juvenile grass carp muscle, implying that Cu-promoted myofiber development might be associated with a decrease in MSTN mRNA levels.
In summary, proper Cu level promoted myofiber growth and development in fish muscle and might be influenced by myoblast proliferation and differentiation.

High level of Cu adversely affected the growth performance and muscle growth of fish
Compared to the optimal level group, the high Cu level (8.07 mg/ kg) reduced growth performance and some of muscle collagen synthesis (except for Smad2 and Smad4 mRNA levels) and myofiber growth and development-related indices of juvenile grass carp, although these results were still higher than those of the control group without added Cu.These results may be partially explained by several aspects.First, our research revealed that high Cu levels reduced growth performance compared to optimal dietary Cu level, which may be related to the reduced FE in the fish.High Cu could reduce the FE of fish, thus leading to reduced growth performance (Wang et al., 2016).Second, the reduction in muscle collagen synthesis by high Cu level in juvenile grass carp may be connected to certain Cu-dependent enzymes.This study found that serum Cp activity was reduced when dietary Cu was too high.Cp is involved in iron metabolism by having iron oxidase activity, converting Fe 2þ to Fe 3þ (Roeser et al., 1970).The reduction of its activity caused iron deposition (Vulpe et al., 1999), and excessive iron content caused downregulation of TGF-b1 mRNA levels (Guo et al., 2017).This might result in the downregulation of signaling molecules that regulate collagen transcription, ultimately leading to a decrease in collagen transcription-related indices.Third, the decrease in myofiber growth and development indices caused by high Cu level may be connected to selenium deficiency.One study revealed that high Cu levels resulted in lower whole-body selenium content in Atlantic salmon (Berntssen et al., 1999), and dietary selenium deficiency decreased the growth and development of rainbow trout myofibers (Wang et al., 2021).More research should be conducted regarding the specific mechanism.

Requirements and the comparison between CuCit and CuSO 4 •5H 2 O of fish
In accordance with the quadratic regression analysis of PWG, serum Cp and collagen content in juvenile grass carp muscle, Cu requirements (CuCit as Cu source) were estimated at 4.74, 4.37 and 4.62 mg/kg diet, respectively (Fig. 4).These results revealed that the Cu requirements of several indicators were comparable.This maybe because many Cu-dependent enzymes use Cu as a cofactor or variable component in fish, which should be given at relatively low quantities to balance nutritional requirements.Additionally, earlier research in our lab on the requirements for other trace elements, such as the requirements for iron based on hematological characteristics (serum iron and hemoglobin), was also comparable to the requirements of juvenile grass carp for growth (Zhang et al., 2016).These findings imply that trace elements for fish health should be utilized with caution.
In addition, the efficacy of CuCit against CuSO 4 $5H 2 O were 131.80% and 115.38% based on PWG and serum Cp activity in juvenile grass carp muscle, respectively.These findings revealed that CuCit was superior to CuSO 4 $5H 2 O.A possible mechanism may be connected to different absorption rates and salt ions between CuCit and CuSO 4 $5H 2 O.The molecules of organic Cu are electrically neutral and are not subject to charge in the intestinal tract, which does not form absorption resistance or deposition phenomena, resulting in rapid absorption and the reduction of many biochemical processes, saving physical energy expenditure and high bioefficiency (Ashmead, 1991).

Conclusion
In conclusion, the present study revealed that dietary Cu increased growth performance and improved the nutritional status of Cu in fish tissues.Furthermore, we first found that appropriate dietary Cu level (4.06 mg/kg) regulates muscle growth and development, which might be related to: (1) elevating collagen content, which might be attributed to the activation of the CTGF/TGF-b1/ Smads signaling pathway, upregulating of LARP6 mRNA levels, and enhancement of the activities of related enzymes (PHD, LH and LOX) for collagen synthesis; (2) promoting proliferation and differentiation of myogenic cells, leading to myofiber growth and development.Finally, based on PWG, serum Cp activity and collagen content quadratic regression analysis in juvenile grass carp muscle, the Cu requirements (CuCit as Cu source) were identified as 4.74, 4.37 and 4.62 mg/kg diet, respectively.Based on PWG and serum Cp activity in juvenile grass carp muscle, compared to CuSO 4 $5H 2 O, the efficacy of CuCit were 131.80% and 115.38%, respectively.
Fig. 1A, the hepatopancreas, serum and muscle Cu contents increased linearly with increasing Cu levels, which considerably increased at the 8.07 mg/kg Cu level in comparison to the Cudeficient group (P < 0.05).Fig. 1B depicts the effects of Cu on

Fig. 2 .
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 200Â ; scale bar ¼ 100 mm).Collagen was stained by red color (n ¼ 3).(B) The proportion of collagen fibers according to statistical analysis of the Sirius red staining (n ¼ 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.Col1a1 ¼ type I collagen a1; Col1a2 ¼ type I collagen a2; LARP6 ¼ La Ribonucleoprotein 6; Sp1 ¼ specificity protein 1; CTGF ¼ connective tissue growth factor; TGF-b1 ¼ transforming growth factor-b1.(HeI) 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 multiplerange tests, P < 0.05.The asterisk (*) indicates a significant difference between 0.99 mg/kg CuCit and CuSO 4 $5H 2 O groups (P < 0.05); the number sign ( # ) indicates a significant difference between 4.06 mg/kg CuCit and CuSO 4 $5H 2 O groups (P < 0.05).Values are means ± SD, n ¼ 3 (for 3 replicate groups, 2 fish per replicate).

Fig. 3 .
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 200Â , scale bar ¼ 100 mm) of muscle in juvenile grass carp supplemented with different Cu levels for 9 weeks visualized using H&E staining.(B) Myofiber diameters (n ¼ 3).(C) Frequency of distribution (%) of the diameters of myofiber (n ¼ 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 CuSO 4 $5H 2 O groups (P < 0.05); the number sign ( # ) indicates a significant difference between 4.06 mg/kg CuCit and CuSO 4 $5H 2 O groups (P < 0.05).Values are means ± SD, n ¼ 3 (for 3 replicate groups, 2 fish per replicate).

Fig. 4 .
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.

Table 1
Ingredients and nutrient composition of the basal diet (air-dry basis, g/kg).

Table 2
Index formulas for growth performance of juvenile grass carp (Ctenopharyngodon idella).

Table 3
Effects of diets containing graded levels of Cu on the growth performance of juvenile grass carp (Ctenopharyngodon idella).

Table 4
Effects of diets containing graded levels of Cu on the proximate composition and physicochemical properties of the muscle of juvenile grass carp (Ctenopharyngodon idella).

Table 5
Correlation coefficient of collagen synthesis and myofiber growth parameters of juvenile grass carp (Ctenopharyngodon idella).