Guanidinoacetic acid improves flesh quality associated with collagen synthesis and nucleotide metabolism in gibel carp CAS V (Carassius gibelio, CAS V)

Abstract

As a precursor to creatine, guanidinoacetic acid (GAA) is widely recognized to enhance growth performance and flesh quality of animals, but the underlying molecular mechanism remains unclear. This study evaluated the effects of dietary GAA supplementation on growth performance, textural properties, and flavor constituents in gibel carp CAS V (Carassius gibelio, CAS V). A total of 300 healthy gibel carp (5.01 ± 0.13 g) at 42 d of age were assigned randomly to 12 tanks (3 replicates per group, 25 fish per replicate). Fish were fed with a basal diet supplemented with graded levels of GAA (0.00, 0.03%, 0.06%, and 0.12%) for 10 weeks. Results demonstrated that GAA supplementation significantly enhanced the specific growth rate (SGR) and crude protein content in gibel carp (P < 0.05). It also enhanced muscle physicochemical attributes by increasing water holding capacity and hardness, along with boosting glycogen content and free glutamate level (P < 0.05). Mechanistically, GAA promoted myofiber development by upregulating protein and gene expression of myogenic regulatory factors (MRFs), leading to increase myofiber density and a higher frequency of myofibers with diameters between 20 and 40 μm (P < 0.05). Furthermore, GAA facilitated collagen synthesis to improve muscle hardness by activating the transforming growth factor-beta 1 (TGF-β1)/mothers against decapentaplegic homolog (SMADs) signaling pathway, upregulating transcript levels of tgf-β1, col1α1, col1α2, and smad3 (P < 0.05). Additionally, GAA increased inosine monophosphate (IMP) content in muscle (P = 0.015), which was associated with enhanced expression of AMPD1 protein and upregulation of the adsl, pkm, and ckm genes (P < 0.05). In conclusion, dietary GAA supplementation enhanced flesh quality of gibel carp via improving growth performance, nutrient deposition, texture characteristics, and flavor components.

1. Introduction

Global fisheries and aquaculture production reached a new record in 2022, surpassing 200 million tons. Notably, aquaculture production exceeded capture fisheries for the first time (FAO, 2024). However, to meet the growing demand for aquatic products, the rapid development of intensive aquaculture has unintentionally brought some negative effects on the quality of fish flesh, such as muscle proximate nutrition declined (Ni et al., 2021), physical properties degradation (Wang et al., 2024), flavor substances reduced (Mu et al., 2022), and off-odors substances increased (Noguera et al., 2024). Muscle quality in fish is a comprehensive trait encompassing proximate composition, fatty acid composition, texture, water-holding capacity, and flavor (Peng et al., 2024). The target of rapamycin (TOR)/ribosomal protein S6 kinase 1 (S6K1) signaling pathway is a key regulator of protein synthesis (Xiao et al., 2023). Guanidinoacetic acid (GAA), a creatine precursor, undergoes enzymatic conversion to creatine in fish muscle via the action of guanidinoacetate methyltransferase (GAMT). The growth performance and muscle crude protein content of grass carp (Ctenopharyngodon idellus) were markedly improved by adding GAA into a fishmeal-free diet (Yang et al., 2021). However, the role of GAA in regulating the protein synthesis signaling pathway in fish muscle is not yet fully understood.

As the main edible component of fish, skeletal muscle is closely associated with the proliferation and hypertrophy of muscle fibers. In grass carp, an optimal dietary protein level improved muscle hardness by increasing myofiber density and collagen synthesis (Dong et al., 2022). Similar to mammals, type I collagen is the most abundant collagen isoform in fish muscle, with its content regulated by synthesis and degradation processes (Wen et al., 2023). A previous study on grass carp showed that GAA increased muscle collagen deposition (Yang et al., 2021).

Alongside physicochemical properties, flavor is one of the most important quality attributes of meat. As a representative flavor nucleotide, inosine monophosphate (IMP) can markedly improve the umami taste of meat and has been employed as a key indicator of meat freshness (Hong et al., 2017). The deposition of IMP is a multifactorial process, influenced by genetic background, feed composition, rearing environment, and muscle type (Huang et al., 2020). A recent study revealed that glutamate upregulates transcript levels of key genes in the de novo synthesis pathway of IMP, and promotes adenosine triphosphate (ATP) production by enhancing energy metabolism, ultimately increasing IMP deposition in muscle (Zuo et al., 2025). As the precursor of creatine, GAA plays a critical regulatory role in facilitating ATP cycling and accelerating energy metabolism (Villasante et al., 2023). Thus, dietary supplementation with GAA might enhance IMP deposition by regulating energy metabolism.

Gibel carp CAS V (Carassius gibelio, CAS V), an important economic fish extensively farmed in China for its rapid growth rate, strong adaptability, and acclaimed muscle quality (Gui, 2024; Yu et al., 2025). However, the flesh texture and flavor substances of farmed fish are easily compromised compared to wild fish (Wang et al., 2024). Although limited studies indicate that GAA can regulate the growth performance and nutrient deposition in aquatic animals, the effects on muscle quality remain unclear (Wuertz and Reiser, 2023). This study investigated the regulatory mechanism of GAA on flesh quality in gibel carp, thereby providing a nutritional strategy for muscle quality regulation.

2. Materials and methods

2.1. Animal ethics statement

All experimental protocols used in this research were examined and authorized by the Institute of Hydrobiology, Chinese Academy of Sciences (approval ID: IHB20140724).

2.2. Experimental design and diets

Table 1 provides the ingredients and nutrient levels of experimental diets. The purity of GAA exceeds 97%, and purchased from Shanghai Aladdin Bio-Technology Co., Ltd. (Shanghai, China). The dietary GAA supplementation levels were 0.00 (Con), 0.03% (0.03GAA), 0.06% (0.06GAA), and 0.12% (0.12GAA). Based on previous researches on the nutritional requirements of gibel carp, four diets with isonitrogenous (33% crude protein) and isolipidic (6.5% crude lipid) were designed (Gao et al., 2019; Yu et al., 2025). Diet components were initially ground and filtered using a 100-mesh sieve, then mixed thoroughly after adding appropriate amount of water. The pellets (diameter: 1 mm) were processed using a single screw pelleting mill (SLP-45, Fishery Machinery and Instrument Research Institute, Chinese Academy of Fishery Sciences, Shanghai, China), followed by oven-dried at 65 °C. They were then preserved under refrigeration at 4 °C until further use.

Table 1.

Ingredients and nutrient levels of experimental diets (%, dry matter basis).

Items	Treatments1
	Con	0.03GAA	0.06GAA	0.12GAA
Ingredients
Fish meal	2.00	2.00	2.00	2.00
Soybean meal	22.00	22.00	22.00	22.00
Rapeseed meal	16.00	16.00	16.00	16.00
Cottonseed protein concentrate	14.00	14.00	14.00	14.00
Wheat flour	24.00	24.00	24.00	24.00
Fish oil	3.00	3.00	3.00	3.00
Soybean oil	3.00	3.00	3.00	3.00
Mineral premix2	5.00	5.00	5.00	5.00
Vitamin premix3	0.39	0.39	0.39	0.39
Carboxymethyl cellulose	3.00	3.00	3.00	3.00
Choline chloride	0.11	0.11	0.11	0.11
Ca(H2PO4)2	1.00	1.00	1.00	1.00
Guanidinoacetic acid	0.00	0.03	0.06	0.12
Cellulose	5.10	5.07	5.04	4.98
L-Lysine	1.00	1.00	1.00	1.00
L-Methionine	0.40	0.40	0.40	0.40
Total	100.00	100.00	100.00	100.00
Nutrient levels4
Crude protein	33.07	33.11	33.38	33.35
Crude lipid	6.72	6.51	6.67	6.55
Dry matter	89.93	89.91	89.66	90.61
Organic matter	82.61	82.52	82.21	83.03
Gross energy, MJ/kg	20.06	20.08	20.13	19.82

¹Con: control diet; 0.03GAA: control diet supplemented with 0.03% GAA; 0.06GAA: control diet supplemented with 0.06% GAA; 0.12GAA: control diet supplemented with 0.12% GAA.

²Mineral premix (mg/kg diet): MgSO₄, 3978.3; FeSO₄, 1250.0; ZnSO₄ 99.8; C₆H₁₀CaO₆·5H₂O, 1750.0; NaH₂PO₄·2H₂O, 12,500.0; NaCl, 500.0; KH₂PO₄, 16,000.0; Ca(H₂PO₄)2·H₂O, 7650.5; MnSO₄, 54.9; CuSO₄, 9.9; KI, 1.5; CoSO₄·7H₂O, 0.9; NaSeO₃, 0.60; corn starch, 899.7.

³Vitamin premix (mg/kg diet): pyridoxine, 20; riboflavin, 20; thiamin, 20; calcium pantothenate, 50; niacin, 100; vitamin C, 100; vitamin E, 50; inositol, 100; vitamin A, 11; vitamin K₃, 10; folic acid, 5; vitamin B₁₂, 0.02; vitamin D₃, 2; biotin, 0.1; cellulose, 3522.

⁴All nutrient levels were analyzed values, except for organic matter.

2.3. Experimental fish and rearing conditions

The juvenile gibel carp CAS V were supplied by Huangshi Fu'er Aquatic Seedlings Co., Ltd. (Huangshi, Hubei, China). All fish were maintained in circular fiberglass tanks (water volume: 1060 L) for 2 weeks to acclimatize the rearing conditions. Following acclimation, the fish were fasted for 24 h, then 300 healthy juvenile gibel carp (mean weight 5.01 ± 0.13 g) were randomly divided into 12 tanks (225 L water volume) in triplicate (25 fish per tank). During the 10-week experiment, all fish were fed corresponding feeds three times daily (08:30, 13:30, and 18:30) to apparent satiation. The water temperature was 30.1 ± 1.2 °C, dissolved oxygen > 6.5 mg/L, pH was 7.0 to 8.0. Ammonia nitrogen concentration was maintained below 0.1 mg/L. A 12-h light/dark cycle was employed with light from 08:00 to 20:00, controlled by an automatic timer.

2.4. Sample collection

Following a 10-week growth experiment, all fish were subjected to a 24-h fasting protocol. Subsequently, fish from each tank were gently removed to count and weight. To evaluate whole-body proximate composition, two fish per tank were randomly sampled for analysis. Six fish per tank were subjected to anesthesia using tricaine methanesulfonate (60 mg/L; Sigma–Aldrich, Inc., St. Louis, MO, USA), and two individuals were quickly dissected on the ice to obtain dorsal white muscle, and immediately frozen in liquid nitrogen, then transferred to a −80 °C freezer for further analysis. The viscera and liver of two fish were separated and weighted to calculate the viscerosomatic index and hepatosomatic index, one part of muscle was used to determine pH value and centrifugal loss, while another (0.3 cm ✕ 0.3 cm ✕ 0.3 cm) was placed in 4% paraformaldehyde for analysis of muscle fiber characteristics. The remaining two fish were used for muscle texture measurement.

2.5. Biochemical analysis

The proximate composition of the diets, whole body, and muscle was evaluated following the guidelines of the AOAC (2005). Specifically, dry matter (DM) content was assessed by drying the sample to constant weight at 105 °C (method 934.01). Crude protein and crude lipid levels were determined through the Kjeldahl procedure (method 984.13) and Soxhlet technique (method 920.39). Crude ash content was determined using a muffle furnace (KSY-12D-16A, Yingshan Jianli Electric Furnace Manufacturing Co., Ltd., Yingshan, Hubei, China) according to standard procedures (method 942.05). Organic matter content was calculated by subtracting ash content from DM. Gross energy was measured using an adiabatic oxygen bomb calorimeter (Parr 6200, Parr Instrument Co., Moline, IL, USA) according to method 983.11. The glycogen (Cat. No. A043-1-1), lactate (Cat. No. A019-2-1), and hydroxyproline (Cat. No. A030-2-1) contents in muscle were measured using commercial kits from Jiancheng Biotech Co., Ltd. (Nanjing, Jiangsu, China) through spectrophotometric after homogenization and centrifugation. Muscle collagen content was calculated by multiplying the hydroxyproline level by 8 (Zeng et al., 2025). The activities of lactate dehydrogenase (LDH; Cat. No. A020-2-2), 5′-nucleotidase (5′-NT; Cat. No. A059-2-2), alkaline phosphatase (AKP; Cat. No. A060-2-2), and acid phosphatase (ACP; Cat. No. A041-2-1) in muscle were assessed using kits provided by Nanjing Jiancheng Bioengineering Institute (Nanjing, Jiangsu, China). Enzymatic levels of cathepsin B (Cat. No. KT24284) and cathepsin L (Cat. No. KT21287) in muscle were assessed using ELISA kits (MSKbio Co., Ltd., Wuhan, Hubei, China).

2.6. Flavor substance content analysis

The free amino acid composition of the dorsal muscle was analyzed using an amino acid analyzer (A300, membraPure GmbH, Bodenheim, Germany), following the method described by Cai et al. (2023). Fatty acid extraction from muscle was conducted using the previously described protocol (Fei et al., 2020). The quantitative analysis of fatty acid profiles in muscle was conducted through a gas chromatography-mass spectrometer (7890A, Agilent Technologies, Inc., Santa Clara, CA, USA), with experimental data presented as the percentage of total fatty acids. Dorsal muscle nucleotides levels were determined using the procedure reported before (Dong et al., 2023). In brief, the fresh samples were thoroughly homogenized with pre-cooled 0.1 mol/L perchloric acid under ice-bath condition. After centrifugation at 1800 × g for 10 min at 4 °C, the resulting supernatant was collected and neutralized to pH 6.4 using KOH. High-performance liquid chromatography (HPLC; Waters e2695, Waters Corporation, Milford, MA, USA) was employed to quantify the concentrations of guanosine monophosphate (GMP), adenosine monophosphate (AMP), and IMP. The mobile phase was prepared by mixing methanol and potassium dihydrogen phosphate (98:2, v/v) and delivered at a flow rate of 1.0 mL/min. Detection was performed using a UV detector at 254 nm.

2.7. Histomorphology

The hematoxylin-eosin (H&E) staining of muscle was conducted following the protocol reported by Yu et al. (2025). After fixation in 4% paraformaldehyde for 24 h, muscle specimens were dehydrated, embedded in paraffin, sectioned at 5 μm, and stained. Images were acquired using an automatic digital slide scanner (Aperio VERSA 8, Leica Biosystems GmbH, Wetzlar, Germany). Muscle fiber number and area of the fiber counting fields were measured by Image J software (National Institutes of Health, Bethesda, MD, USA). Muscle fiber density was calculated using the previously described method (Valente et al., 2016). Myofiber diameters were calculated using the formula;

$$S=\pi r^2,$$

where S and r represent muscle fiber cross-sectional area and radius, respectively. The frequency distribution of myofiber diameter was then calculated. Six samples were taken from each group, and more than 100 muscle fibers per sample were measured.

To analyze the effects of GAA on collagen and glycogen accumulation in gibel carp muscle, periodic acid-Schiff stain (PAS) and Masson staining were performed by Servicebio Co., Ltd. (Wuhan, Hubei, China). Muscle images were acquired using the method mentioned earlier. Collagen and glycogen contents were quantified using ImageJ (National Institutes of Health, Bethesda, MD, USA) following the established protocols by Cai et al. (2023). Six samples per experimental group were measured, with results expressed as relative area percentages.

2.8. Texture analysis

The muscle texture of gibel carp was determined through a texture analyzer (TA.XT plusC, Stable Micro Systems Ltd., Godalming, Surrey, UK) following an established experimental protocol from previous research (Dong et al., 2023). Briefly, a probe was pressed down at a rate of 1.00 mm/s on the muscle, and the trigger force was 20.0 g, pressed down 3 mm after touching the fish fillet and maintained for 30 s. The probe was then returned to its original height to continue with a second downward pressure at a compression ratio of 60%. Hardness, resilience, flexibility, springiness, and toughness were measured in this research. The centrifugal loss and cooking loss of muscle were detected based on previous study (Li et al., 2019). One gram of white muscle was sampled and homogenized with 10 mL distilled water for pH_{0 h} and pH_{24 h} measurement using a pH meter (FE20, Mettler-Toledo Instruments Co., Ltd., Shanghai, China). The fish samples were stored in a refrigerator maintained at 4 °C during this period.

2.9. Quantitative real-time PCR analysis

The RNA extraction, cDNA synthesis, and quantitative real-time PCR protocols were conducted based on previous procedures in our laboratory (Cai et al., 2023). RNA integrity was assessed by 1% agarose gel electrophoresis, followed by measurement of RNA concentration using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA, USA). First-strand cDNA was synthesized using M-MLV reverse transcriptase (Cat. No. 28025013, Invitrogen, Thermo Fisher Scientific Inc., Carlsbad, CA, USA). Quantitative real-time PCR was performed following standardized protocols. Table S1 lists the primers used in this investigation, gapdh served as an internal reference. The full names of the abbreviations of all genes can be found in supplementary file Abbreviations and Full Names of Genes and Proteins. The relative expression levels were determined following Pfaffl (2001).

2.10. Western blotting

The western blotting procedure was adopted using the previously described method (Wu et al., 2023). Total muscle protein was extracted using radio immunoprecipitation assay (RIPA) lysis buffer (Cat. No. P0013B, Beyotime Biotechnology Co., Ltd., Shanghai, China), which contained both protease and phosphatase inhibitors. The homogenate was centrifuged, and protein concentration of supernatant was subsequently quantified employing a BCA assay kit (Cat. No. P0010S, Beyotime Biotechnology Co., Ltd., Shanghai, China). Objective proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride (PVDF) membranes. Following blocking with 5% skim milk, the PVDF membrane was incubated with primary antibody overnight at 4 °C. Subsequently, the membrane was washed and incubated with a secondary antibody. Protein bands were detected using enhanced chemiluminescence, and images were acquired with an ImageQuant LAS 4000 mini (Cytiva Life Sciences, Uppsala, Sweden). ImageJ software (National Institutes of Health, Bethesda, MD, USA) was used to quantify the pertinent proteins. Antibody information is shown in Table S2. The full names of the abbreviations of all proteins can be found in supplementary file Abbreviations and Full Names of Genes and Proteins.

2.11. Statistical analyses

Table S3 provides the calculation formulas for growth parameters. Data processing was carried out employing SPSS 25.0 software (SPSS Inc., Chicago, IL, USA), one-way analysis of variance (ANOVA) and Duncan's multiple range test were used to evaluate the differences in mean values across different groups, under the assumptions of normality and homogeneity of variances. The statistical model as follows:

$$Y_{ij}=\mu +{\alpha }_i+{\epsilon }_{ij}\text{,}$$

where Y_ij represents the dependent variables; μ represents the overall mean; α_i represents the group difference; and ε_ij represents the random error component.

To identify potential trends, the linear and quadratic effects of diets with different GAA levels on all measured parameters were evaluated via statistical modeling. The linear regression model is as follows:

$$y={\beta }_0+{\beta }_{1}x+\epsilon \text{,}$$

the quadratic regression model is as follows:

$$y={\beta }_0+{\beta }_{1}x+{\beta }_2{x}^2+\epsilon \text{,}$$

where y represents the dependent variable; β₀ represents the constant (intercept); x represents the independent variable; β₁ and β₂ represent the regression coefficients; ε represents the random error term.Values are shown as mean and standard error of the mean (SEM). P ＜ 0.05 was regarded as statistically significant.

3. Results

3.1. Growth performance

Following a 10-week feeding experiment, fish growth parameters, organ indices, and proximate composition were determined. Following dietary supplementation with GAA, the weight gain rate (WGR) and specific growth rate (SGR) were significantly elevated (P < 0.05; Table 2). In terms of feed utilization, GAA had no significant effect on feed conversion ratio (FCR) of gibel carp (P > 0.05). Additionally, no significant differences were detected in hepatosomatic index (HSI), viscerosomatic index (VSI), condition factor (CF), and carcass ratio (CR) of gibel carp among different treatments (P > 0.05). However, dietary supplementation of GAA significantly increased the body length (BL) of gibel carp (P = 0.003). The contents of moisture and crude lipid of whole body did not show any statistical differences among treatments (P > 0.05). Nevertheless, the whole body crude protein and ash contents were significantly improved following GAA supplementation, especially in the 0.12GAA group (P < 0.05). No significant difference in dorsal muscle composition was observed among the treatments (P > 0.05).

Table 2.

Effects of GAA on growth performance, organ indices, whole body composition and muscle composition of gibel carp.

Items	Treatments1				SEM	P-value
	Con	0.03GAA	0.06GAA	0.12GAA		ANOVA	Linear	Quadratic
IBW, g	5.15	4.94	4.92	5.04	0.038	0.096	0.404	0.024
FBW, g	27.08	28.23	29.26	29.25	0.417	0.202	0.071	0.262
WGR, %	425.67b	471.91ab	494.34a	480.74a	9.935	0.046	0.038	0.034
SGR, %/d	2.37b	2.49a	2.54a	2.51a	0.025	0.041	0.033	0.033
FI, g/fish	33.95	32.20	35.16	34.40	0.568	0.333	0.441	0.977
FCR	1.58	1.39	1.50	1.47	0.033	0.251	0.488	0.319
HSI, %	3.59	3.49	3.96	3.96	0.163	0.654	0.316	0.859
VSI, %	7.55	8.88	8.90	9.04	0.284	0.212	0.109	0.218
CF, g/cm3	3.05	3.19	3.10	3.21	0.027	0.098	0.085	0.775
CR, %	66.27	64.96	65.47	67.30	0.499	0.383	0.301	0.183
BL, mm	96.44b	100.44a	101.67a	99.44a	0.551	0.003	0.075	0.001
Whole body composition, % of wet weight
Moisture	69.05	68.73	68.32	66.88	0.380	0.190	0.042	0.667
Crude protein	16.64b	17.56ab	17.83a	18.06a	0.208	0.047	0.014	0.147
Crude lipid	8.54	8.58	8.63	9.14	0.297	0.912	0.523	0.801
Ash	3.73b	4.01ab	4.00ab	4.31a	0.080	0.047	0.010	0.804
Muscle composition, % of wet weight
Moisture	77.35	77.05	77.02	77.23	0.099	0.672	0.856	0.253
Crude protein	18.56	18.56	18.71	18.56	0.097	0.949	0.941	0.672
Crude lipid	2.69	3.01	2.97	2.92	0.105	0.756	0.616	0.423

GAA = guanidinoacetic acid; IBM = initial body weight; FBW = final body weight; WGR = weight gain rate; SGR = specific growth rate; FI = feed intake; FCR = feed conversion ratio; HSI = hepatosomatic index; VSI = viscerosomatic index; CF = condition factor; CR = carcass ratio; BL = body length; SEM = standard error of the mean.

Values marked with different superscript letters within individual rows demonstrate statistically significant differences (P < 0.05). Data are expressed as means and SEM (n = 3).

¹Con: control diet; 0.03GAA: control diet supplemented with 0.03% GAA; 0.06GAA: control diet supplemented with 0.06% GAA; 0.12GAA: control diet supplemented with 0.12% GAA.

3.2. Muscle amino acids and fatty acids composition

The compositional characteristics of muscle free amino acid were displayed in Table 3. Compared with the Con group, the concentrations of Glu, Val, Phe, and Arg in muscle were significantly elevated with GAA supplementation (P < 0.05), and peaked at 0.06GAA group. Methionine was found to decrease initially and then increase with GAA supplementation (P_quadratic = 0.023), among which the content in the 0.12GAA group was significantly elevated compared with 0.06GAA group (P = 0.045). Moreover, supplementation with GAA significantly reduced the concentration of His in muscle (P = 0.015). Nevertheless, dietary GAA supplementation resulted in higher levels of umami amino acids in muscle, particularly in the 0.06GAA group (P = 0.043).

Table 3.

Effects of GAA on muscle free amino acid composition (μg/g) of gibel carp.

Items	Treatments1				SEM	P-value
	Con	0.03GAA	0.06GAA	0.12GAA		ANOVA	Linear	Quadratic
Asp	5.44	5.89	6.20	8.25	0.741	0.578	0.183	0.747
Thr	259.97	247.34	255.91	239.19	7.168	0.767	0.397	0.911
Ser	64.30	60.95	56.53	60.49	2.510	0.776	0.612	0.395
Glu	173.35b	202.81ab	257.87a	181.32b	12.160	0.048	0.792	0.010
Gly	135.38	125.67	140.66	129.92	10.057	0.963	0.954	0.909
Ala	194.06ab	250.58a	241.52a	167.07b	12.447	0.042	0.174	0.013
Val	6.86b	5.97b	11.42a	4.59b	0.886	0.026	0.471	0.029
Met	8.09ab	6.51ab	4.05b	11.75a	1.132	0.045	0.100	0.023
Ile	11.77	10.83	10.19	8.39	0.901	0.627	0.198	0.982
Leu	20.68	19.27	23.38	16.38	1.426	0.391	0.357	0.300
Tyr	2.73	2.30	2.48	3.92	0.521	0.717	0.371	0.479
Phe	2.18b	2.03b	4.83a	1.65b	0.364	0.002	0.791	0.002
His	3635.37a	3098.49b	3098.64b	3019.55b	81.218	0.015	0.011	0.059
Lys	162.23	165.91	168.48	129.18	13.417	0.731	0.371	0.514
Arg	311.03bc	367.65b	468.55a	259.77c	21.753	0.001	0.215	<0.001
Pro	99.46	117.65	143.72	108.69	9.436	0.403	0.747	0.119
Sweet amino acids2	753.17	802.19	838.34	705.36	23.923	0.226	0.378	0.065
Umami amino acids3	178.79b	208.70ab	264.07a	189.57b	11.940	0.043	0.713	0.009

GAA = guanidinoacetic acid; SEM = standard error of the mean.

Values marked with different superscript letters within individual rows demonstrate statistically significant differences (P < 0.05). Data are expressed as means and SEM (n = 6).

¹Con: control diet; 0.03GAA: control diet supplemented with 0.03% GAA; 0.06GAA: control diet supplemented with 0.06% GAA; 0.12GAA: control diet supplemented with 0.12% GAA.

²Sweet amino acids = Gly + Ser + Thr + Pro + Ala.

³Umami amino acids = Glu + Asp.

The muscle fatty acid profile of gibel carp is presented in Table 4. A noticeable decrease in the concentration of C22:0 was observed in the 0.12GAA group (P = 0.050). Similarly, following GAA supplementation, the level of C24:1n9 was significantly reduced (P = 0.048). Furthermore, GAA had no significant effect on other fatty acids (P > 0.05).

Table 4.

Effects of GAA on fatty acid profile (% of total fatty acid methyl esters) in muscle of gibel carp.

Items	Treatments1				SEM	P-value
	Con	0.03GAA	0.06GAA	0.12GAA		ANOVA	Linear	Quadratic
C14:0	1.62	1.61	1.57	1.61	0.025	0.868	0.901	0.464
C15:0	0.37	0.36	0.34	0.35	0.086	0.482	0.316	0.261
C16:0	20.90	21.15	20.67	19.41	0.417	0.473	0.156	0.542
C16:1	3.03	2.40	3.08	2.98	0.137	0.253	0.574	0.632
C17:0	0.44	0.41	0.37	0.41	0.013	0.426	0.566	0.141
C17:1	0.39	0.36	0.35	0.38	0.091	0.253	0.959	0.051
C18:0	6.04	6.38	5.79	5.67	0.143	0.311	0.175	0.772
C18:1n9c	27.76	28.13	28.14	28.59	0.423	0.938	0.545	0.977
C18:2n6	22.70	22.56	21.72	23.60	0.356	0.312	0.344	0.149
C18:3n6	0.26	0.27	0.28	0.25	0.005	0.217	0.699	0.053
C20:0	0.20	0.20	0.18	0.18	0.005	0.391	0.165	0.513
C18:3n3	1.63	1.73	1.70	1.82	0.040	0.476	0.160	0.957
C20:1	1.44	1.40	1.52	1.51	0.037	0.694	0.410	0.895
C20:2	0.54	0.49	0.48	0.51	0.015	0.560	0.564	0.205
C20:3n6	0.89	0.90	0.92	0.87	0.019	0.860	0.761	0.466
C22:0	0.23a	0.20ab	0.22a	0.18b	0.008	0.050	0.029	0.579
C20:3n3	0.67	0.68	0.75	0.66	0.025	0.583	0.911	0.252
C22:1n	0.29	0.28	0.28	0.23	0.019	0.709	0.299	0.660
C20:5n3	1.39	1.57	1.57	1.51	0.062	0.759	0.697	0.350
C23:0	0.17	0.17	0.17	0.16	0.005	0.936	0.554	0.839
C24:1n9	0.40a	0.30b	0.26b	0.29b	0.019	0.048	0.068	0.027
C22:6n3	8.68	8.39	9.57	8.73	0.525	0.884	0.874	0.674
ΣSFA	30.06	30.55	29.38	28.07	0.549	0.418	0.132	0.654
ΣMUFA	33.04	32.86	33.62	33.97	0.473	0.868	0.490	0.878
ΣPUFA	39.70	36.58	37.00	37.96	0.800	0.937	0.553	0.842

GAA = guanidinoacetic acid; SFA = saturated fatty acid; MUFA = monounsaturated fatty acid; PUFA = polyunsaturated fatty acid; SEM = standard error of the mean.

Values marked with different superscript letters within individual rows demonstrate statistically significant differences (P < 0.05). Data are expressed as means and SEM (n = 6).

¹Con: control diet; 0.03GAA: control diet supplemented with 0.03% GAA; 0.06GAA: control diet supplemented with 0.06% GAA; 0.12GAA: control diet supplemented with 0.12% GAA.

3.3. Muscle physicochemical properties

As Table 5 depicted, the centrifugal loss and cooking loss were gradually decreased with the addition of GAA, compared to the Con group, both 0.06GAA and 0.12GAA significantly reduced centrifugal loss, while 0.12GAA significantly decreased cooking loss (P < 0.05). The pH_{0 h} and pH_{24 h} of muscle were not significantly influenced by GAA (P > 0.05). Similarly, the content of lactic acid and activity of LDH of muscle did not show any significantly difference among treatments (P > 0.05). However, compared with the Con group, muscle hardness and flexibility were significantly improved in 0.12GAA group (P < 0.05). Besides, no markedly changes in resilience, rupture strength, springiness, or toughness of muscle were observed when GAA was added to the diet (P > 0.05).

Table 5.

Effects of GAA on muscle physicochemical properties of gibel carp.

Items	Treatments1				SEM	P-value
	Con	0.03GAA	0.06GAA	0.12GAA		ANOVA	Linear	Quadratic
Centrifugal loss, %	18.50a	15.81ab	14.64b	14.01b	0.606	0.032	0.009	0.157
Cooking loss, %	20.52a	19.49ab	18.57ab	17.11b	0.445	0.034	0.004	0.784
pH0 h	6.95	7.00	6.94	7.03	0.015	0.139	0.130	0.492
pH24 h	6.87	7.00	6.91	7.00	0.041	0.624	0.438	0.805
Lactic acid, mmol/g prot	1.56	1.66	1.49	1.67	0.068	0.775	0.695	0.681
Lactate dehydrogenase, U/g prot	21,067.70	22,549.78	20,209.41	22,470.97	546.944	0.377	0.539	0.512
Hardness, N	0.70b	0.70b	0.75ab	0.85a	0.021	0.034	0.005	0.525
Resilience, %	31.25	32.88	35.02	33.84	1.103	0.696	0.422	0.413
Rupture strength, N	1.06	1.11	1.18	0.97	0.042	0.363	0.404	0.138
Flexibility, g/s	17.12b	16.90b	18.71ab	21.86a	0.733	0.049	0.008	0.471
Springiness, %	44.40	46.33	45.93	40.02	0.973	0.074	0.049	0.070
Toughness, N·s	4.10ab	4.34a	4.75a	3.54b	0.139	0.009	0.069	0.005

GAA = guanidinoacetic acid; SEM = standard error of the mean.

Values marked with different superscript letters within individual rows demonstrate statistically significant differences (P < 0.05). Data are expressed as means and SEM (n = 6).

¹Con: control diet; 0.03GAA: control diet supplemented with 0.03% GAA; 0.06GAA: control diet supplemented with 0.06% GAA; 0.12GAA: control diet supplemented with 0.12% GAA.

3.4. Muscle nucleotide metabolism

The effects of GAA on muscle nucleotide metabolism are shown in Table 6 and Fig. 1. The concentration of IMP reached peak in the 0.03GAA group (P = 0.015), although GAA supplementation had no significant effect on muscle AMP and GMP contents (Table 6; P > 0.05). Similarly, no statistically significant variations were detected in the activities of 5′-NT, ACP, and AKP among treatment groups (P > 0.05). Moreover, the expression of genes associated with IMP de novo synthesis pathway, including ppat, atic, gart, and paics were not altered between different groups (Fig. 1A; P > 0.05). However, the expression levels of genes involved in IMP transformation pathways were affected by GAA supplementation (Fig. 1B). The transcript abundance of adsl, ampd1, pkm, and ckm were significantly increased following GAA supplementation (P < 0.05). Furthermore, protein expression level of AMPD1 was significantly improved in 0.03GAA group (Fig. 1C; P = 0.050). The transcriptional activity of the impdh2 gene elevated significantly in the 0.06GAA group (P = 0.008).

Table 6.

Effects of GAA on muscle nucleotide deposition of gibel carp.

Items	Treatments1				SEM	P-value
	Con	0.03GAA	0.06GAA	0.12GAA		ANOVA	Linear	Quadratic
IMP, μg/g	2273.66ab	2478.55a	2257.46ab	2112.58b	42.993	0.015	0.023	0.107
GMP, μg/g	449.89	381.80	491.63	877.49	89.121	0.194	0.055	0.336
AMP, μg/g	175.12	149.28	166.94	166.06	8.088	0.744	0.952	0.561
ACP, U/g prot	12.36	11.28	13.16	13.48	0.585	0.579	0.330	0.818
AKP, U/g prot	5.85	6.27	6.32	5.78	0.378	0.947	0.878	0.568
5′-NT, U/g prot	3.69	4.30	5.28	4.46	0.327	0.411	0.398	0.178

GAA = guanidinoacetic acid; IMP = inosine monophosphate; GMP = guanosine monophosphate; AMP = adenosine monophosphate; ACP = acid phosphatases; AKP = alkaline phosphatases; 5′-NT = 5′-nucleotidase; SEM = standard error of the mean.

Values marked with different superscript letters within individual rows demonstrate statistically significant differences (P < 0.05). Data are expressed as means and SEM (n = 6).

¹Con: control diet; 0.03GAA: control diet supplemented with 0.03% GAA; 0.06GAA: control diet supplemented with 0.06% GAA; 0.12GAA: control diet supplemented with 0.12% GAA.

Fig. 1.

Effects of GAA on muscle nucleotide metabolism of gibel carp. (A and B) The relative expression levels of genes associated with nucleotide metabolism. (C) Expression of AMPD1 was evaluated using western blotting. Con: control diet; 0.03GAA: control diet supplemented with 0.03% GAA; 0.06GAA: control diet supplemented with 0.06% GAA; 0.12GAA: control diet supplemented with 0.12% GAA. Results are presented as means and standard error of the mean (n = 6), distinct letters designate statistically significant differences (P < 0.05). GAA = guanidinoacetic acid.

3.5. Muscle glycogen and protein synthesis

As shown in Fig. 2, periodic acid-Schiff staining was used to detect the effect of GAA levels on glycogen deposition in gibel carp (Fig. 2A). Semiquantitative analysis indicated that 0.12% GAA significantly improved muscle glycogen level (P < 0.001). A similar result was observed from the glycogen content assay using a commercial kit (Fig. 2B). Furthermore, 0.12% GAA significantly enhanced the relative expression levels of pgm5, ugp2, and gys genes, which are key regulatory elements in glycogen synthesis (P < 0.05). The expression levels of genes associated with protein synthesis are shown in Fig. 2C. The relative expression levels of mtor and s6k1 genes in muscle were significantly improved in 0.06GAA group (P < 0.05).

Fig. 2.

Effects of GAA on muscle glycogen and protein metabolism of gibel carp. (A) Periodic acid-Schiff staining of fish muscle (200×), glycogen was stained by purple (n = 6). (B) Glycogen content and relative expression levels of glycogen metabolism genes. (C) Relative expression levels of genes related to protein synthesis. Con: control diet; 0.03GAA: control diet supplemented with 0.03% GAA; 0.06GAA: control diet supplemented with 0.06% GAA; 0.12GAA: control diet supplemented with 0.12% GAA. Results are presented as means and standard error of the mean (n = 6), distinct letters designate statistically significant differences (P < 0.05). GAA = guanidinoacetic acid.

3.6. Myofiber characteristics

The effects of GAA on the muscle myofiber characteristics of gibel carp are shown in Fig. 3 and Table 7. GAA supplementation significantly increased the muscle fiber density and diminished the average muscle fiber diameter (Table 7; P < 0.05). Besides, 0.06% GAA significantly improved the frequency distribution of myofibers with diameters less than 20 μm (P = 0.012). Dietary supplementation with GAA significantly increased the myofiber diameter frequency distribution in the 20 to 40 μm range (P = 0.001). Conversely, GAA significantly reduced the frequency distribution of myofibers with diameters between 80 and 100 μm and greater than 100 μm (P < 0.05). Moreover, the protein expression levels of PGC1α, MYOD, and MYOG were notably increased and peaked in the 0.03GAA group (Fig. 3B; P < 0.05). Correspondingly, following GAA supplementation, the relative expression levels of myod and myog genes were notably upregulated (Fig. 3C; P < 0.05). After GAA supplementation, the relative expression levels of cyclin D, myhc, mylc, and mrf4 genes were notably elevated (P < 0.05). Conversely, the relative expression level of mstn gene was significantly reduced following dietary supplementation with GAA (P = 0.036).

Fig. 3.

Effects of GAA on muscle histological and myofiber growth in gibel carp. (A) Hematoxylin-eosin staining in cross-sections of fish muscle (200×; n = 6). (B) Protein expression levels of PGC1α, MYOD, and MYOG. (C) The relative expression levels of genes related to myoblast proliferation and differentiation. Con: control diet; 0.03GAA: control diet supplemented with 0.03% GAA; 0.06GAA: control diet supplemented with 0.06% GAA; 0.12GAA: control diet supplemented with 0.12% GAA. Results are presented as means and standard error of the mean (n = 6), distinct letters designate statistically significant differences (P ≤ 0.05). GAA = guanidinoacetic acid.

Table 7.

Effects of GAA on muscle histology of gibel carp.

Items	Treatments1				SEM	P-value
	Con	0.03GAA	0.06GAA	0.12GAA		ANOVA	Linear	Quadratic
Muscle fiber density, fibers/mm2	190.63b	263.19a	263.54a	253.47a	9.151	0.004	0.021	0.005
Average diameter, μm	68.49a	57.61b	55.64b	55.07b	1.272	<0.001	<0.001	<0.001
Frequency of myofibers (< 20 μm), %	1.31b	3.46ab	5.78a	2.32b	0.539	0.012	0.502	0.002
Frequency of myofibers (20–40 μm), %	11.76b	24.77a	23.31a	23.93a	1.514	0.001	0.005	0.006
Frequency of myofibers (40–60 μm), %	27.43	24.49	28.84	32.89	1.190	0.080	0.031	0.313
Frequency of myofibers (60–80 μm), %	26.31	28.14	26.90	27.49	0.765	0.869	0.765	0.765
Frequency of myofibers (80–100 μm), %	21.83a	17.45ab	14.14b	11.96b	1.176	0.008	0.001	0.221
Frequency of myofibers (> 100 μm), %	11.36a	1.69b	1.04b	1.56b	0.954	<0.001	<0.001	<0.001

GAA = guanidinoacetic acid; SEM = standard error of the mean.

Values marked with different superscript letters within individual rows demonstrate statistically significant differences (P < 0.05). Data are expressed as means and SEM (n = 6).

¹Con: control diet; 0.03GAA: control diet supplemented with 0.03% GAA; 0.06GAA: control diet supplemented with 0.06% GAA; 0.12GAA: control diet supplemented with 0.12% GAA.

3.7. Expression of genes and proteins involved in collagen biosynthesis

The effects of GAA on collagen deposition in gibel carp are shown in Fig. 4 and Table 8. Collagen accumulation in muscle was assessed using Masson staining (Fig. 4A). The results showed that collagen fiber area (blue) elevated remarkably in the GAA-supplemented groups, reaching a maximum at 0.12GAA group (P = 0.001). Additionally, muscle hydroxyproline level and collagen content were significantly elevated in the 0.12GAA group (Table 8; P < 0.05). Although GAA had no effect on cathepsin B activity, 0.12GAA significantly improved the activity of cathepsin L (P = 0.028). Furthermore, supplementation with GAA significantly upregulated protein levels of TGF-β1, p-SMAD2, and SMAD4 in muscle (Fig. 4B; P < 0.05). Accordingly, the relative expression levels of tgf-β1, colα1, colα2, and smad3 genes were significantly elevated in the 0.12GAA group (Fig. 4C; P < 0.05).

Fig. 4.

Effects of GAA on collagen content in gibel carp muscle. (A) Masson's trichrome stain of muscle and area of collagen fibers (200 × ; n = 6). (B) Protein expression levels of TGF-β1, p-SMAD2, SMAD2, and SMAD4 in muscle and corresponding quantification. (C) The relative expression levels of genes involved in collagen synthesis. Con: control diet; 0.03GAA: control diet supplemented with 0.03% GAA; 0.06GAA: control diet supplemented with 0.06% GAA; 0.12GAA: control diet supplemented with 0.12% GAA. Results are presented as means and standard error of the mean (n = 6), distinct letters designate statistically significant differences (P < 0.05). GAA = guanidinoacetic acid.

Table 8.

Effects of GAA on muscle collagen deposition of gibel carp.

Items	Treatments1				SEM	P-value
	Con	0.03GAA	0.06GAA	0.12GAA		ANOVA	Linear	Quadratic
Hydroxyproline content, mg/g	0.25b	0.20b	0.31ab	0.38a	0.022	0.017	0.006	0.398
Collagen content, mg/g	2.04b	1.62b	2.49ab	3.07a	0.179	0.017	0.006	0.398
Cathepsin L, U/g prot	2.03b	2.15ab	1.89b	2.36a	0.060	0.028	0.060	0.088
Cathepsin B, U/g prot	7.09	6.47	6.43	6.19	0.193	0.420	0.149	0.496

GAA = guanidinoacetic acid; SEM = standard error of the mean.

Values marked with different superscript letters within individual rows demonstrate statistically significant differences (P < 0.05). Data are expressed as means and SEM (n = 6).

¹Con: control diet; 0.03GAA: control diet supplemented with 0.03% GAA; 0.06GAA: control diet supplemented with 0.06% GAA; 0.12GAA: control diet supplemented with 0.12% GAA.

4. Discussion

4.1. Dietary GAA supplementation improved growth performance and nutritional composition of gibel carp

Previous researches have demonstrated that appropriate creatine intake has a growth-promoting effect in fish (Wuertz and Reiser, 2023). Guanidinoacetic acid, a precursor of creatine, can be converted into creatine in fish muscle through guanidinoacetate methyltransferase. Research on Nile tilapia (Oreochromis niloticus) and grass carp have demonstrated the growth-promoting effects of GAA at dietary inclusion levels of 150 to 600 mg/kg and 0.06%–0.18%, respectively (Aziza et al., 2020; Yang et al., 2021). Similarly, the present study indicates that GAA enhances growth performance in juvenile gibel carp. In contrast, supplementation with 0.5% or 1% GAA did not improve growth in juvenile red drum (Sciaenops ocellatus) (Stites et al., 2020), which due to higher supplementation levels of GAA, aligns with findings in bullfrogs, where 0.4 g/kg GAA significantly promoted growth, but further increases led to a notable decline in growth performance (Zeng et al., 2018).

The improved growth performance was accompanied by enhanced nutritional quality in gibel carp fed GAA-supplemented diets. In this study, dietary supplementation with GAA has been shown to enhance whole-body protein deposition. This finding aligns with previous observations in grass carp (Yang et al., 2021). The regulation of muscle protein deposition is governed by the dynamic between protein synthesis and degradation. As a principal regulatory network, the mechanistic target of rapamycin (mTOR) signaling pathway plays a central role in regulating protein synthesis by promoting the phosphorylation of S6K1 (Fuentes et al., 2013). Guanidinoacetic acid serves as the direct precursor for creatine synthesis, and its supplementation accelerates ATP regeneration through elevating intramuscular creatine stores (Villasante et al., 2023). Adequate ATP availability supports the activation of the mTOR pathway, thereby promoting muscle protein deposition. Dietary supplementation of 8.48 g/kg creatine elevated the phosphorylation levels of TOR, 4EBP1, and S6K1, thereby improving protein deposition in grass carp (Tian et al., 2023). In this research, dietary supplementation with 0.06% GAA significantly improved the relative expression levels of mtor and s6k1 genes. This suggests that GAA may enhance protein synthesis by modulating the mTOR signaling pathway. Furthermore, glycogen serves as a crucial energy storage compound in fish muscle, and its content and metabolic processes significantly contribute to flesh quality and nutritional value (Liu et al., 2020). This study found that 0.12% GAA distinctively improved the muscle glycogen content in gibel carp. The present finding aligns with a previous investigation that documented significant changes in glycogen levels in bullfrog (Lithobates catesbeiana) following GAA supplementation (Zeng et al., 2018). During glycogen synthesis, gys, pgm5, and ugp2 coordinate essential enzymatic steps that mediate the conversion of glucose to glycogen, thereby regulating glycogen accumulation (Zhang et al., 2021). Earlier research in gibel carp found that ribose improved the muscle glycogen deposition through enhancing the transcript level of gys (Cai et al., 2023). In Nile tilapia, a dose of 5000 mg/kg uridine upregulated the expression of ugp2 mRNA, leading to higher glycogen accumulation (Zhou et al., 2023). In this research, GAA greatly elevated the relative expression levels of gys, pgm5, and ugp2 genes. These results indicate that the elevated muscle glycogen level may be attributed to the promoting effect of GAA on glycogen synthesis.

4.2. Dietary GAA supplementation may improve muscle physicochemical properties by regulating muscle growth and collagen metabolism

The sensory experience of meat products is primarily determined by nutritional and physical properties of muscle (Matarneh et al., 2021). The present study demonstrated that dietary supplementation of 0.12% GAA greatly improved the hardness and flexibility of muscle. The effect of GAA on muscle hardness is likely related to collagen content and myofiber characteristics. Specifically, a positive correlation between collagen content and muscle hardness has been demonstrated in several fish species (Wen et al., 2023). The present results showed that 0.12% GAA remarkably elevated hydroxyproline content and collagen fiber in juvenile gibel carp, as evidenced by Masson's trichrome staining, indicating that GAA improved the collagen content of fish muscle. Type I collagen, the predominant collagen in fish muscle, col1α1 and col1α2 peptide chains. Its synthesis is regulated by the transforming growth factor-beta 1 (TGF-β1)/mothers against decapentaplegic homolog (SMADs) signaling pathway. TGF-β1 phosphorylates SMAD2 and SMAD3, which can bind to SMAD4 to form a transcription factor, thereby enhancing the expression of genes involved in collagen synthesis (Jinnin, 2010). In this research, GAA supplementation improved the protein expression of TGF-β1, p-SMAD2, and SMAD4, as well as the relative expression levels of tgf-β1, col1α1, col1α2, and smad3 genes. These results indicate that GAA promotes collagen synthesis through activating the TGF-β1/SMADs signaling pathway. Additionally, it was reported that collagen degradation and its dynamic balance are influenced by cathepsin L and cathepsin B (Wang et al., 2015). The investigation revealed that GAA supplementation had no significant effect on muscle cathepsin B activity, whereas 0.12% GAA increased cathepsin L activity. Beyond the established role of collagen, increased muscle hardness may also be related to muscle fiber characteristics. It has been revealed that muscle hardness is significantly positive correlated with muscle fiber density in most teleost fishes (Dong et al., 2022). In line with previous study (Yang et al., 2021), GAA supplementation remarkably elevated myofiber density while reducing the average myofiber diameter in our study. These results indicate that GAA promotes myofiber proliferation and attenuates hypertrophy. Peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1α) functions as a transcriptional coactivator that significantly upregulates myod and myog transcription, which are key regulators of myogenesis (Lin et al., 2014). The current study found that GAA supplementation improved protein expression levels of PGC-1α, MYOG, and MYOD, along with the relative expression levels of myhc, mylc, mrf4, myod, and myog genes. As a negative regulator of muscle fiber development, mstn knockout promotes myofiber hypertrophy and upregulates the transcription of myod, myog, and myf5 (Yeh et al., 2017). Analysis revealed that mstn relative expression level was notably decreased following GAA supplementation. Collectively, these results suggest that GAA may improve muscle hardness by promoting myofiber development and collagen deposition.

While myofiber structure directly affects textural attributes, other physicochemical characteristics of muscle, including pH and water-holding capacity, are also critical in determining overall sensory quality. Typically, the pH of fish muscle decreases initially and then increases with extended storage time (Li et al., 2015). During this process, muscle glycogen is converted to lactate acid and H⁺ ions via the anaerobic glycolytic pathway. Lactate dehydrogenase, a key enzyme in glycolysis pathway that reduces pyruvate to lactate, and the decline in pH is primarily attributed to lactate accumulation. Correlation analysis suggested that vitamin A improved the post-mortem pH of grass carp by reducing lactate concentration (Wu et al., 2022). In the present study, although 0.12% GAA increased muscle glycogen content, no notable differences were observed in pH, lactic acid concentration, or LDH activity in fish muscle. These findings align with previous reports on spotted seabass (Lateolabrax maculatus) (Lin et al., 2023). A similar pattern was observed in grass carp, where histidine increased muscle glycogen content and glycolytic flux but reduced lactate accumulation and delayed pH decline, which possibly related to enhanced H⁺ ion transport (Zeng et al., 2025). In addition, water-holding capacity is also one of the most important parameters of muscle quality and is closely linked to muscle pH. Generally, the lower of centrifugal loss or cooking loss, indicating greater water-holding capacity of muscle. In this experiment, both centrifugal loss and cooking loss in muscle were gradually decreased following dietary GAA supplementation. This indicates that GAA effectively improves the water-holding capacity of muscle.

4.3. Dietary GAA supplementation improved the flesh flavor of gibel carp

Free amino acids are important flavor compounds in fish. They are mainly classified as umami, sweetness, and bitterness based on their unique taste. A previous study revealed that optimal tryptophan supplementation notably increased the content of free aspartic acid and glutamate, thereby improving the umami flavor of grass carp flesh (Xiao et al., 2023). In this study, supplementation with 0.06% GAA markedly improved the free glutamate and umami amino acids contents. Besides, as key flavor precursors, fatty acids have a significant impact on the formation of flesh flavor. A recent study illustrated that α-lipoic acid elevated the eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and polyunsaturated fatty acid (PUFA) contents of muscle through regulating fatty acid synthesis, consequently improving the flesh flavor of grass carp (Zhang et al., 2025). In this study, GAA supplementation showed no significant effect on monounsaturated fatty acid (MUFA) or PUFA content, but reduced levels of C22:0 and C24:1n9. However, another study reported that dietary supplementation with GAA improved the omega-3 polyunsaturated fatty acid (n-3 PUFA), MUFA, EPA, and DHA contents in grass carp muscle (Yang et al., 2021). This discrepancy with the present findings may be attributed to differences in fish species or dietary composition, warranting further in-depth research.

Flavor nucleotides are crucial components of flesh flavor and greatly contribute to the umami taste. In fish muscle, IMP is the predominant flavor nucleotide, which can enhance the flavor of the meat several-fold when combined with glutamate (Mouritsen and Khandelia, 2012). In vivo, there are three main pathways for IMP synthesis: de novo synthesis, salvage pathway, and transformation pathway (Huang et al., 2020). In the de novo pathway, phosphoribosyl pyrophosphate amidotransferase (PPAT) serves as the first rate-limiting enzyme, regulating IMP synthesis by modulating downstream genes expression. A recent study revealed that knocking down ppat remarkably reduced the transcription levels of ppat, paics, adsl, and atic, thereby decreasing IMP deposition (Zuo et al., 2025). In present study, no significant changes were observed in the relative expression levels of ppat, gart, paics, and atic genes. These findings suggest that the regulatory role of GAA on IMP deposition in gibel carp may not be mediated by the de novo synthesis pathway. Furthermore, the transformation pathway critically regulates IMP deposition. Adenosine monophosphate deaminase 1 (AMPD1), the rate-limiting enzyme of this pathway, catalyzes the conversion of AMP to IMP (He et al., 2024). Previous studies have shown that the adsl, ckm, and pkm genes can indirectly promote IMP deposition by accelerating ATP metabolism (Huang et al., 2023). In present research, supplementing with GAA upregulated AMPD1 protein expression and increased relative expression levels of ampd1, adsl, ckm, and pkm genes. Conversely, the hydrolysis of IMP is regulated by ACP, AKP, and 5′-NT. In present study, the activities of ACP, AKP, and 5′-NT in muscle remained unchanged following GAA supplementation. These results suggest that GAA may improve IMP synthesis by enhancing the transformation pathway.

5. Conclusion

In this study, dietary supplementation with GAA improved the growth and flesh quality of gibel carp. The improved muscle nutritional value is likely attributable to enhanced the capacity for protein and glycogen synthesis. GAA further improved muscle physicochemical characteristics and sensory quality, potentially through promoting myofiber growth and development, a process regulated by PGC1α, MYOD, and MYOG. The improvement in muscle hardness may be linked to enhanced collagen biosynthesis, which is regulated through the TGF-β1/SMADs signaling pathway. Furthermore, GAA-mediated improvement of muscle flavor quality may be associated with increased free amino acids and IMP deposition. In summary, these results demonstrate that GAA supplementation is a viable strategy to enhance growth performance and flesh quality of gibel carp.

Credit Author Statement

Yu Wang: Writing – original draft, Investigation, Formal analysis. Yan Zhang: Investigation, Data curation. Liyun Wu: Methodology. Chaoyue Li: Validation, Software. Qiaozhen Chen: Validation, Software. Dong Han: Methodology. Haokun Liu: Methodology. Zhimin Zhang: Methodology. Shouqi Xie: Supervision, Funding acquisition. Junyan Jin: Writing – review & editing, Funding acquisition.

Declaration of competing interest

We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, and there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the content of this paper.

Appendix A. Supplementary data

The following is the Supplementary data to this article.