Dietary L-carnosine enhances the growth, muscle hardness and protein deposition in red swamp crayfish (Procambarus clarkii)

Abstract

The relatively low edible muscle yield and inconsistent flesh texture of red swamp crayfish limit its commercial value and highlight the need for effective nutritional interventions. Although L-carnosine has shown beneficial effects on muscle growth and quality in vertebrates, its nutritional functions and underlying mechanisms in crustaceans remain unclear. This study aimed to evaluate the effects of dietary L-carnosine on growth performance and muscle quality in red swamp crayfish (Procambarus clarkii). A total of 324 red swamp crayfish, with an initial body weight of 6.19 ± 0.03 g, were randomly divided into 6 groups of three replicates and 54 crayfish per group (18 crayfish per replicate). Six experimental diets containing graded levels of L-carnosine (3.35, 59.28, 106.84, 222.20, 452.93, and 821.16 mg/kg) were fed for 8 weeks. Regression analysis revealed dose-dependent responses in multiple parameters. Weight gain rate, specific growth rate, protein efficiency ratio, protein deposition rate, muscle percentage, whole-body and muscle crude protein content, muscle textural properties (hardness and chewiness), hydroxyproline levels (alkaline-insoluble and total), myofiber density, small myofiber proportion (<50 μm), and total amino acid content exhibited quadratic trends (P < 0.05), with peak values observed in the 452.93 mg/kg group. Conversely, feed conversion ratio and large myofiber proportion (>70 μm) showed significant quadratic decreases (P < 0.05), reaching their lowest values at 452.93 mg/kg. Transcriptional analysis demonstrated that, compared with the 3.35 mg/kg L-carnosine group, the 452.93 mg/kg L-carnosine group significantly regulated genes involved in several key pathways: mTORC1 signaling (igf-1, pi3k, akt, tor, s6k1, and 4ebp1), eIF2B-eIF2 signaling (eif2a), ubiquitin-proteasome system (ub, psma2, psmc1, and murf1), autophagy-lysosomal system (atg16l1, atg5, beclin1, and atg12), TGFβ/Smads pathway (TGF-β1, smad6, smad3, smad4, and colla1), and myogenic regulators (mef2a, mef2b, and mstn) (P < 0.05). These molecular modulations were consistent with observed phenotypic improvements. Overall, the findings demonstrated that a dietary L-carnosine level of 458.06 to 500.00 mg/kg, as determined by quadratic regression analysis, significantly enhances growth performance, feed utilization, muscle hardness and chewiness, and nutritive value in red swamp crayfish.

1. Introduction

Consumer preference and market value for aquatic products are fundamentally linked to the biological mechanisms governing muscular development (Snyder et al., 2012). Unlike mammals, which primarily rely on myofiber hypertrophy for muscle growth, aquatic species exhibit muscle accretion through both hypertrophy and hyperplasia (cellular proliferation) (Jagot et al., 2018). Consequently, skeletal muscle accretion in aquatic species can be enhanced by elevating myofiber density and improving protein retention efficiency (Wilschut et al., 2010). However, nutrient utilization limits often restrict protein deposition, with less than 30% of ingested dietary protein retained in muscle (NCR, 2011). Animal-derived proteins (including fish meal, krill meal, etc.) generally exhibit superior digestibility and deposition than plant-based sources in aquafeeds (Daniel, 2018; Jiang et al., 2013; Liu et al., 2024; Xu et al., 2024). Nevertheless, rising costs and supply instability of marine- and poultry-derived proteins have driven increased incorporation of plant proteins in diets (Macusi et al., 2023), which often compromises growth performance and muscle quality (Valente et al., 2016; Wu et al., 2021; Zhang et al., 2023). Strategic supplementation with marine-derived bioactive compounds (including taurine, carnosine, creatine, etc.) may therefore provide a synergistic means of improving both yield and muscle attributes.

L-Carnosine, a dipeptide naturally abundant in animal tissues, is increasingly recognized in aquaculture as a functional nutrient lacking in plant-based diets (Boldyrev et al., 2013; Iovine et al., 2014; Li and Wu, 2020). It exhibits antioxidant activity and influences myofiber type conversion, as evidenced in terrestrial livestock (Cong et al., 2017; Liu et al., 2022; Zhao et al., 2020). Poultry studies further indicate that dietary L-carnosine enhances muscle texture, oxidative stability, and fiber-type transition (Cong et al., 2017; Hu et al., 2009). In fish, L-carnosine (50–500 mg/kg) primarily functions as an antioxidant, improves feed efficiency in certain species, enhances muscle quality and texture, and regulates lipid metabolism. For instance, juvenile turbot (Scophthalmus maximus) exhibited enhanced feed conversion and hepatic antioxidant capacity at 50 mg/kg (Wang et al., 2017). In orange-spotted grouper (Epinephelus coioides), supplementation with L-carnosine at 195.14 mg/kg significantly improved growth, muscle antioxidant capacity, and flesh quality (Li et al., 2024). For largemouth bass (Micropterus salmoides) fed a high-lipid diet, supplementation with L-carnitine at 200 mg/kg improved growth, antioxidant ability, and lipid metabolism (Chen et al., 2020), and dietary L-carnosine at 500 mg/kg improved muscle texture, regulated lipid metabolism, and enhanced nutritional value under high-fat dietary conditions (Wang et al., 2022). In addition, dietary supplementation of L-carnosine precursors can modulate the endogenous L-carnosine pool in vertebrates (Bauchart et al., 2007; Kai et al., 2015; Tomonaga et al., 2012). Despite these findings, the role of L-carnosine in muscle protein deposition, collagen synthesis, and growth performance remains largely unexplored in invertebrates.

With the highest overall economic output in China's freshwater aquaculture, the red swamp crayfish (Procambarus clarkii) industry plays a crucial role in meeting the demand for high-quality aquatic products. According to the China Fisheries Statistical Yearbook 2025, red swamp crayfish production reached 3.45 million tons, ranking fourth among China's freshwater aquaculture species (Bureau of Fisheries of the MARA of the People's Republic of China, 2025). Nevertheless, its comparatively lower muscle percentage and higher proportion of smaller-sized individuals remain inconsistent with current market requirements. To address these production challenges, juvenile red swamp crayfish (initial body weight: 6.19 ± 0.03 g) were fed six diets containing graded L-carnosine levels for 8 weeks to determine the optimal dietary L-carnosine inclusion level using dose–response analysis (Tian et al., 2023). In addition, the underlying mechanisms by which L-carnosine affects muscle quality, particularly in relation to protein deposition and collagen synthesis, were investigated. This systematic study provides valuable insights into the nutritional application of L-carnosine for improving production efficiency and flesh quality in red swamp crayfish.

2. Materials and methods

2.1. Animal ethics statement

Red swamp crayfish (Procambarus clarkii) are legally permitted for aquaculture in China. All authors complied with applicable guidelines for the care and use of experimental animals. All animal handling and experimental procedures were approved by the Institutional Animal Care and Use Committee of Yangtze River Fisheries Research Institute (according to YFI, 2023TJ01 of March 6, 2023).

2.2. Experimental diets formulation

The control diet was developed with protein components (fish meal, rapeseed meal, soybean meal, cottonseed protein, and peanut meal), and lipid components (fish oil, soybean oil, and soybean lecithin), with detailed formulations and nutritional content outlined in Table 1. Dietary supplementation was achieved by adding L-carnosine (≥99% purity, Shanghai Yuanye Bio-Technology Co., Ltd., Shanghai, China) at six graded concentrations: 0 (control group), 50, 100, 200, 400, and 800 mg/kg to the basal formulation. Bentonite was used as a filler to balance the formulations, ensuring all diets had identical total weight. The raw materials were pulverized, sieved through a 0.3-mm mesh, and accurately weighed. Precision blending was performed using stepwise expansion methodology with complete lipid and water integration. Extrusion processing was conducted using a twin-screw extruder (F-26, Guangzhou Huagong Guangdian Technology Co., Ltd., Guangzhou, Guangdong, China) to produce 2.0-mm diameter strands. These strands were subsequently cured (90 °C, 20 min) in a sealed oven and dried (60 °C, 2 h) in a DW belt dryer (Changzhou Suzheng Drying Equipment Co., Ltd., Changzhou, Jiangsu, China). Final products were size-standardized and stored at −20 °C for further use. The actual L-carnosine content in the feed was determined by liquid chromatography-mass spectrometry (LC-MS) analysis to be 3.35, 59.28, 106.84, 222.20, 452.93, and 821.16 mg/kg, respectively. The analysis was conducted by Shanghai Sensichip Infotech Co., Ltd. (Shanghai, China).

Table 1.

Composition of the experimental diets (dry matter basis, g/kg).

Items	Dietary L-carnosine levels, mg/kg
	3.35	59.28	106.84	222.20	452.93	821.16
Ingredients
Fish meal	50.00	50.00	50.00	50.00	50.00	50.00
Soybean meal	160.00	160.00	160.00	160.00	160.00	160.00
Wheat flour	250.00	250.00	250.00	250.00	250.00	250.00
Rapeseed meal	150.00	150.00	150.00	150.00	150.00	150.00
Cottonseed meal	40.00	40.00	40.00	40.00	40.00	40.00
Peanut meal	150.00	150.00	150.00	150.00	150.00	150.00
Sodium alginate	20.00	20.00	20.00	20.00	20.00	20.00
Ca(H2PO4)2	23.00	23.00	23.00	23.00	23.00	23.00
Yeast extract	20.00	20.00	20.00	20.00	20.00	20.00
Fish oil	22.00	22.00	22.00	22.00	22.00	22.00
Soybean oil	22.00	22.00	22.00	22.00	22.00	22.00
Bentonite	44.00	43.95	43.90	43.80	43.60	43.20
L-Carnosine	0.00	0.05	0.10	0.20	0.40	0.80
Soybean lecithin	10.00	10.00	10.00	10.00	10.00	10.00
Vitamin premix1	10.00	10.00	10.00	10.00	10.00	10.00
Cholesterol	5.00	5.00	5.00	5.00	5.00	5.00
Mineral premix2	10.00	10.00	10.00	10.00	10.00	10.00
Vitamin C	3.00	3.00	3.00	3.00	3.00	3.00
Choline chloride	2.00	2.00	2.00	2.00	2.00	2.00
Chitosan	1.00	1.00	1.00	1.00	1.00	1.00
Astaxanthin	0.40	0.40	0.40	0.40	0.40	0.40
L-Threonine	3.60	3.60	3.60	3.60	3.60	3.60
L-Lysine	3.00	3.00	3.00	3.00	3.00	3.00
Methionine–methionine	1.00	1.00	1.00	1.00	1.00	1.00
Total	1000.00	1000.00	1000.00	1000.00	1000.00	1000.00
Nutrients3
Dry matter	953.96	952.55	952.48	953.44	951.72	954.34
Crude protein	370.60	370.87	371.03	371.37	371.58	371.90
Crude fat	66.65	66.81	66.76	66.43	66.82	66.49
Organic matter	880.34	880.62	880.93	880.59	880.60	880.93
Crude fiber	72.23	72.11	72.07	72.01	72.00	72.31
Nitrogen-free extract	324.82	323.38	323.55	324.22	321.92	324.57
L-Carnosine, mg/kg	3.35	59.28	106.84	222.20	452.93	821.16
Gross energy, KJ/g	19.63	19.52	19.27	19.30	19.30	19.60

¹Per kg of vitamin premix contains vitamin A 4 g, vitamin D 0.02 g, vitamin E 10 g, vitamin K₃ 10 g, vitamin B₁ 10 g, vitamin B₂ 10 g, vitamin B₆ 20 g, nicotinic acid 40 g, biotin 0.2 g, calcium pantothenate 20 g, folic acid 0.5 g, vitamin B₁₂ 0.01 g, vitamin C 20 g, inositol 400 g, all ingredients were diluted with micro-cellulose to 1 kg. These chemical reagents were all purchased from Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China).

²Per kg of mineral premix contains KIO₃ 0.6 g, Na₂SeO₃·5H₂O 0.08 g, KH₂PO₄ 320 g, MgSO₄ 200 g, MnSO₄·H₂O 20 g, CuCl₂·2H₂O 2 g, ZnSO₄·7H₂O 60 g, FeSO₄·7H₂O 50 g, NaCl 100 g, CoCl₂·6H₂O 2 g, all ingredients were diluted with micro-cellulose to 1 kg. These vitamin reagents were all purchased from Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China).

³All values were analyzed.

2.3. Feeding management

The red crayfish were sourced from an aquacultural facility (Qianjiang, Hubei, China). Prior to trial initiation, the red crayfish underwent a 14-d conditioning phase in controlled rearing conditions while receiving the control diet. Ten individuals were randomly selected and cryopreserved (−20 °C) as the initial samples. The culture system consisted of 18 square tanks, each measuring 1 m² in area and 0.35 m in depth. Each tank had a total volume of 350 L, with a working water volume of 250 L. To mitigate aggressive interactions and cannibalism, particularly during the vulnerable post-molt phase, each tank was furnished with 18 shelter tubes, 1 blackout net, and 2 climbing nets. These structures provide essential refuges, increase habitat complexity, disperse individuals, and create a secure environment for molting (Yao et al., 2024). A total of 324 juvenile crayfish (initial mass, 6.19 ± 0.03 g) with intact appendages and similar size were randomly allocated to 18 tanks, with 18 individuals per tank. An 8-week feeding trial was carried out from June 26 to August 20, 2023, at the Yangtze River Fisheries Research Institute, Chinese Academy of Fishery Sciences (Wuhan, Hubei, China). A continuous flow of fresh water was supplied to each tank at a rate of 300 L/h. The water, sourced from the municipal supply, was dechlorinated and aerated prior to use. The water temperature of 25.9 ± 1.4 °C was maintained solely by the ambient conditions of the laboratory, with no active temperature control applied. Daily average temperature data are presented in Table S1. Water quality parameters were carefully regulated, with dissolved oxygenmaintained above 5 mg/L, pH at 8.3 ± 0.1, and ammonia nitrogen concentrations kept below 0.05 mg/L. Each diet was randomly assigned to three tanks. Twice-daily feeding regimes (08:00 and 18:00) were implemented with quantified ration administration. Uneaten diets were collected via siphoning 2 h after feeding, dried and weighed to quantify feed consumption. Dead crayfish were immediately removed and weighed.

2.4. Sample collection

Upon completion of the 8-week trial period, all experimental crayfish underwent a 24 h fasting period, then counted and weighed for calculation of growth performance. A stock solution of clove oil was prepared by diluting it with 96% ethanol at a volume ratio of 1:10. From this stock, a working clove oil solution with a concentration of 350 μL/L (0.035% v/v) was prepared by mixing an appropriate aliquot with water and bringing it to the final volume in a volumetric flask. Prior to sampling, all crayfish were anesthetized by immersion in 350 μL/L clove oil solution. Anesthesia was continued until a deep plane was achieved, as indicated by loss of equilibrium and absence of response to tactile stimulus (Elmas and Karadal, 2022). Three randomly selected crayfish per tank were preserved at −20 °C for whole-body proximate composition analysis. For hemolymph collection, nine specimens per tank were randomly sampled through pericardial cavity puncture using sterile syringes. The collected hemolymph was transferred to pre-chilled 1.5-mL centrifuge tubes and maintained at 4 °C for 4 h prior to high-speed centrifugation (14,400 × g, 4 °C, 20 min). The resulting supernatant was aliquoted and cryopreserved at −80 °C for subsequent biochemical assays. Hepatopancreatic and intestinal tissues were collected immediately after dissection, weighed, and stored at −40 °C for subsequent analysis of hepatosomatic index (HSI) and digestive enzyme activities. To evaluate whether the effects of L-carnosine on digestion are systemic or organ-specific, both tissue types were examined. The hepatopancreas serves as the primary organ for synthesizing and secreting digestive enzymes, whereas the intestine is responsible for terminal nutrient hydrolysis and absorption. Assessing these two tissues together provides integrated insight into digestive function along the gastrointestinal tract, an established approach in crustacean nutrition research (Yao et al., 2024; Tian et al., 2023). Finally, after weighing, the muscle samples (1.0 cm × 1.0 cm × 0.5 cm) were sampled for the determination of muscle texture. The muscle samples (0.5 cm × 0.5 cm × 0.5 cm) were sampled and placed in 4% paraformaldehyde for muscle histological analysis. Residual muscle tissue was partitioned for differential preservation: one aliquot was flash-frozen in liquid nitrogen and archived at −80 °C for gene expression detection, while the parallel aliquot was maintained at −40 °C for comprehensive analyses including antioxidant enzyme activity, collagen quantification, amino acid profiling, and proximate composition determination.

2.5. Determination of indicators and methods

2.5.1. Calculation of growth and feed utilization

$$\text{Weight}\text{gain}\text{rate}\left(\text{WGR},\%\right)=\left(\text{Final}\text{mean}\text{weight}–\text{Initial}\text{mean}\text{weight}\right)/\text{Initial}\text{mean}\text{weight}\times 100\text{;}$$

$$\text{Specific}\text{growth}\text{rate}\left(\text{SGR},\%/\mathrm{d}\right)=\left[\ln \left(\text{Final mean}\text{weight}/\text{Initial}\text{mean}\text{weight}\right)\right]/56\times 100\text{;}$$

$$\text{Feed}\text{conversion}\text{ratio}\left(\text{FCR}\right)=\text{Dry}\text{feed}\text{intake}/\text{Wet}\text{weight}\text{gain;}$$

$$\text{Muscle}\text{percentage}\left(\text{MP},\%\right)=\text{Abdomen}\text{muscle}\text{weight}/\text{Crayfish}\text{body}\text{weight}\times 100\text{;}$$

$$\text{Hepatosomatic}\text{index}\left(\text{HSI},\%\right)=\text{Hepatopancreas}\text{weight}/\text{Crayfish}\text{body}\text{weight}\times 100\text{;}$$

$$\text{Survival}\text{rate}\left(\text{SR},\%\right)=\left(\text{Final}\text{crayfish}\text{number}/\text{Initial}\text{crayfish}\text{number}\right)\times 100\text{;}$$

$$\text{Protein}\text{efficiency}\text{ratio}\left(\text{PER},\%\right)=\text{Weight}\text{gain}/\text{Crude}\text{protein}\text{intake}\times 100\text{;}$$

$$\text{Protein}\text{deposition}\text{rate}\left(\text{PDR},\%\right)=\text{Protein}\text{gain}/\text{Total}\text{protein}\text{consumption}\times 100\text{.}$$

2.5.2. Proximate analysis

The proximate compositions were quantified following AOAC (2023) standard procedures. Moisture content in the experimental diets was measured using the constant weight method at 135 °C (method 930.15), while that of whole crayfish and muscle was determined using a freeze dryer (Christ Beta 2–4 LD plus LT, Marin Christ Corporation, Osterode, Germany). Gross energy of the diets was analyzed using an adiabatic bomb calorimeter (SDC311, Hunan Sunde Technology Co., Ltd., Changsha, Hunan, China) according to ISO 9831:1998 (ISO, 1998). The crude lipid contents of the diets, whole crayfish, and muscle were determined by petroleum ether Soxhlet extraction (Sox 606, Hanon Advanced Technology Group Co., Ltd., China; method 920.39). The crude protein contents of the diets, whole crayfish, and muscle were analyzed by an auto Kjeldahl system (Kjelflex K-360, BUCHI Labortechnik AG, Flawil, Switzerland; method 954.01). The ash contents of the diets, whole crayfish, and muscle were determined by calcination in a muffle furnace (SX-4-10, Hubei Yingshan State-owned Radio Components Factory, Yingshan, Hubei, China) for 10 h at 550 °C (method 942.05). The organic matter (OM) content of the experimental diets was calculated by difference according to the following Eq.:

$$\text{OM}\left(\mathrm{g}/\text{kg}\text{DM}\right)=1000–\text{ash}\left(\mathrm{g}/\text{kg}\text{DM}\right)\text{,}$$

where ash represents the total ash content expressed on a dry matter basis.

2.5.3. Digestive enzymes activities of hepatopancreas and intestine

Fresh hepatopancreas and intestine tissues were precisely weighed and placed into 2-mL centrifuge tubes. Following homogenization with nine volumes of physiological saline using a glass homogenizer (Biaoma FJ-150, Shanghai Specimen and Model Factory, Shanghai, China), the homogenates were centrifuged at 4 °C (654 × g) for 10 min using a centrifuge (5407, Eppendorf AG, Hamburg, Germany). The resulting supernatants were collected and stored at −80 °C for subsequent analysis. Activities of digestive enzymes in the hepatopancreas and intestine were assessed using commercial assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, Jiangsu, China). Total protein (TP; A045–4) content was measured using the bicinchoninic acid (BCA) method, and lipase (LPS) activity was determined using a lipase assay kit (A054-2-1); absorbance for these assays was measured using a microplate reader (ELx800, BioTek Instruments, Inc., Winooski, VT, USA). Total protease activity was determined using the Folin reagent method, and amylase (AMS; C016-1-1) activity was measured using the iodine starch staining method; absorbance for these assays was measured using a visible spectrophotometer (V-1100, Shanghai Macylab Instruments Co., Ltd., Shanghai, China). All procedures were strictly followed according to the manufacturer's protocols.

2.5.4. Determination of muscle antioxidant capacity

Fresh muscle tissue was accurately weighed and transferred into 2-mL centrifuge tubes. Samples were homogenized in nine volumes of physiological saline using a glass homogenizer (Biaoma FJ-150) and subsequently centrifuged at 4 °C (654 × g) for 10 min using a centrifuge (model 5407). The resulting supernatants were collected and stored at −80 °C for analysis. Antioxidant enzyme activities and malondialdehyde (MDA) levels were quantified using commercial kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, Jiangsu, China). Superoxide dismutase (SOD; A001–3) activity was evaluated using the water-soluble tetrazolium-1 (WST-1) method, and absorbance was measured using a microplate reader (ELx800). The MDA (A003–1) content was assessed using the thiobarbituric acid (TBA) method , total antioxidant capacity (T-AOC; A015-2-1) was measured using the colorimetric method and catalase (CAT; A007-1-1) activity was determined using the ammonium molybdate method; absorbance for these assays was measured using a visible spectrophotometer (V-1100). All indices were calculated according to the manufacturer's instructions.

2.5.5. Muscle texture properties analysis

Muscle texture attributes were evaluated via a texture analyzer (TVT-300XP, Perten Instruments [Beijing] Co., Ltd., Beijing, China) equipped with a P-cy5s cylindrical probe. Each sample underwent two compression tests at a data acquisition rate of 200 pps. The experimental protocol adhered to the detailed methodology described by Li et al. (2022).

2.5.6. Determination of muscle collagen content

Fresh muscle tissue was accurately weighed into 50-mL centrifuge tubes. Homogenization was performed with nine volumes of distilled water for 1 min, followed by the addition of 10 mL of pre-cooled 0.2 mol/L sodium hydroxide solution. The mixture as incubated with continuous shaking at 4 °C for 4 h. Subsequent centrifugation at 4 °C (10,000 × g) for 30 min separated alkali-soluble hydroxyproline (supernatant) from alkali-insoluble hydroxyproline (pellet). The alkali-insoluble fraction was transferred to ampoules with 3 mL of 6 mol/L hydrochloric acid for hydrolysis at 110 °C (20 h), then diluted to 10 mL final volume. The hydroxyproline quantification experimental protocol adhered to the detailed methodology described by Zhang et al. (2013). Colorimetric analysis was performed using a spectrophotometer (V-1100, MACYLAB Instrument Co., Ltd., Shanghai, China).

2.5.7. Histological analysis of muscle

Fresh muscle samples were fixed in 4% formaldehyde for 24 h. Through sequential dehydration in a graded ethanol, clearing, and paraffin embedding, tissue blocks were prepared. Transverse sections (5 μm) were obtained using a microtome, deparaffinized, rehydrated, and stained with hematoxylin-eosin (H&E). Samples were first examined under a light microscope (BX53, Olympus Corporation, Tokyo, Japan), followed by quantitative measurement of myofiber diameter and density using Image-J software (1.54p, National Institutes of Health, Bethesda, ML, USA).

2.5.8. Analysis of muscle amino acid content

A precisely weighed portion (0.1000 g) of the freeze-dried muscle sample (mentioned in section 2.4.1) was placed into a glass ampoule containing 12 mL of hydrochloric acid (6 mol/L). The sample was hydrolyzed at 110 °C for 24 h. After cooling, the hydrolysate was filtered and the filtrate was diluted to a final volume of 100 mL with ultrapure water. A 1.5 mL aliquot was vacuum-dried at 60 °C (24 h), reconstituted in 2 mL distilled water, and re-dried for 24 h. The residue was dissolved in 8 mL of 0.02 mol/L hydrochloric acid, filtered through 0.22-μm aqueous-phase membranes, and analyzed via automatic amino acid analyzer (L-8900, Hitachi High-Technologies Corporation, Tokyo, Japan).

2.5.9. Gene mRNA expression levels

Total RNA was extracted from frozen muscle samples using TRIzol reagent (Life Technologies, Carlsbad, CA, USA) in centrifuge tubes. RNA integrity was verified by 1% agarose gel electrophoresis, with concentration measured via microspectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA, USA). Complementary DNA (cDNA) was synthesized from total RNA using the PrimeScript RT Reagent Kit with gDNA Eraser (Perfect Real Time, Takara Biotechnology Co., Ltd., Dalian, Liaoning, China), according to the manufacturer's instructions. Quantitative real-time PCR (QuantStudio6 FLEX Q6, Applied Biosystems, Waltham, MA, USA) with SYBR Premix Ex Taq (Takara Biotechnology Co., Ltd., Dalian, Liaoning, China) amplified target genes. The primers are listed in Table S2. The 18s rRNA was selected as the housekeeping gene. The full names of the abbreviations of all genes can be found in Table S3. Relative expression was calculated by the 2^−ΔΔCt method as described by Livak and Schmittgen (2001). The expression level of each target gene in the control group was set to 1, and the expression levels of genes in other groups were calculated relative to the control group.

2.6. Statistical analysis

All datasets were processed through a parametric statistical framework implemented in GraphPad Prism 8 software (GraphPad Software, Inc., La Jolla, CA, USA). Between-group comparisons were conducted with one-way ANOVA followed by Tukey's HSD post-hoc test, with significance set at P < 0.05. To characterize response patterns, orthogonal polynomial comparison was used to analyze data trends and the adjusted R² values, enabling detection of linear and nonlinear (quadratic) trends. For graphical representation, bar graphs were made using GraphPad Prism 8, while continuous relationships were depicted via regression curves generated in Origin (2019) (OriginLab Inc., Northampton, MA, USA). The statistical model for one-way ANOVA can be expressed as:

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

where Y_ij is the observed value for the dependent variable; μ is the overall mean; α_i is the main effect of the i-th level (i.e., different dietary L-carnosine levels); ε_ij is the random error term, representing the influence of other random factors beyond the independent variable on the dependent variable.

3. Results

3.1. Growth performance

In groups containing 59.28 to 821.16 mg/kg L-carnosine, WGR, SGR, PER, and PDR were significantly higher than in the control group (P < 0.001), with peak values observed at 452.93 mg/kg (Table 2). Similarly, MP was elevated at L-carnosine levels of 106.84 to 821.16 mg/kg (P < 0.001), also reaching its maximum at 452.93 mg/kg. In contrast, the FCR was significantly lower than the control at dietary L-carnosine concentrations of 59.28 to 821.16 mg/kg (P < 0.001). Survival rate and HSI remained unaffected across all treatment levels (P > 0.05). Dietary L-carnosine elicited distinct dose-dependent patterns in red swamp crayfish growth parameters. Based on the quadratic curve regression model analysis for WGR, SGR, MC, PER, PDR, and FCR, the optimal level of dietary L-carnosine was 458.06 to 500.00 mg/kg (Fig. 1).

Table 2.

Effect of dietary L-carnosine levels on growth performance of red swamp crayfish.

Items	Dietary L-carnosine levels, mg/kg						SEM	P-value			Adjusted R2
	3.35	59.28	106.84	222.20	452.93	821.16		ANOVA	Linear	Quadratic	Linear	Quadratic
IBW, g	6.20	6.19	6.17	6.18	6.16	6.21	0.006	0.088	0.712	0.012	−0.053	0.374
FBW, g	14.97d	16.74b	16.71c	18.18b	18.87a	17.07c	0.300	<0.001	0.057	<0.001	0.159	0.924
WGR, %	141.39d	170.31c	170.70c	194.20b	206.45a	174.82c	4.988	<0.001	0.069	<0.001	0.142	0.933
SGR, %/d	1.57d	1.77c	1.78c	1.93b	2.02a	1.88b	0.034	<0.001	0.006	<0.001	0.341	0.924
FCR	1.28a	1.22b	1.18c	1.17c	1.10d	1.20bc	0.013	<0.001	0.066	<0.001	0.145	0.901
MP, %	12.80d	12.99cd	13.24c	13.66b	14.30a	13.21c	0.122	<0.001	0.129	<0.001	0.084	0.921
HSI, %	7.34	7.92	7.65	6.96	7.48	7.43	0.101	0.106	0.639	0.674	−0.048	−0.075
SR, %	87.14	90.48	92.86	92.86	90.48	87.38	1.042	0.469	0.473	0.237	−0.028	0.065
PER, %	205.45d	213.97c	217.26bc	221.84ab	225.89a	214.81b	1.606	<0.001	0.161	<0.001	0.064	0.882
FI, g	179.13b	193.17ab	191.33ab	213.60a	214.93a	187.53ab	3.988	0.016	0.973	0.022	−0.062	0.318
PDR, %	30.82c	33.60b	34.35b	35.75b	39.90a	35.41b	0.685	<0.001	0.020	<0.001	0.251	0.873

IBW = initial mean weight; FBW = final mean weight; WGR = weight gain rate; SGR = specific growth rate; FCR = feed conversion ratio; MP = muscle percentage; HSI = hepatosomatic index; SR = survival rate; PER = protein efficiency ratio; FI = supplement feed intake; PDR = protein deposition rate; SEM = standard error of the mean.

Values with different superscripts in the same row are significantly different (P < 0.05), n = 6.

Fig. 1.

Quadratic curve model analysis of the relation between dietary L-carnosine levels and key growth index of red swamp crayfish. (A) Weight gain rate. (B) Specific growth rate. (C) Muscle percentage. (D) Protein efficiency rate. (E) Protein deposition rate. (F) Feed conversion ratio.

3.2. Proximate composition

The crude protein content in the whole body and muscle exhibited a quadratic trend response to dietary L-carnosine levels, with maximum enhancements observed at 452.93 mg/kg (P < 0.001; Table 3). Compared with the control group, the maximum enhancement in whole body occurred at 452.93 mg/kg (P = 0.005), while muscle also showed significantly higher protein content (P < 0.001). Dietary L-carnosine levels did not significantly affect moisture, crude fat, or ash content in the whole body and muscle composition (P > 0.05).

Table 3.

Effect of dietary L-carnosine levels on whole crayfish and abdominal muscle composition of red swamp crayfish (g/kg, wet mass).

Items	Dietary L-carnosine levels, mg/kg						SEM	P-value			Adjusted R2
	3.35	59.28	106.84	222.20	452.93	821.16		ANOVA	Linear	Quadratic	Linear	Quadratic
Whole body
Moisture	672.40	682.61	683.52	678.18	679.62	677.32	2.574	0.912	0.914	0.887	−0.099	−0.190
Crude protein	136.32b	140.69b	140.42b	147.73ab	158.90a	145.28ab	2.071	0.005	0.060	<0.001	0.153	0.625
Crude lipid	29.69	29.76	29.94	29.81	29.51	29.31	1.084	0.927	0.257	0.527	0.039	−0.060
Ash	51.94	51.96	54.97	52.05	54.69	56.66	0.690	0.189	0.023	0.085	0.361	0.293
Abdominal muscle
Moisture	770.76	774.61	767.02	774.63	774.30	767.65	1.684	0.657	0.619	0.519	−0.046	−0.039
Crude protein	167.48c	173.84bc	177.53abc	178.65ab	185.29a	180.25ab	0.130	<0.001	0.003	<0.001	0.211	0.444
Crude lipid	5.68	5.71	5.60	5.45	5.35	5.37	0.098	0.849	0.217	0.369	0.016	0.002
Ash	13.73	13.65	13.95	13.66	13.20	13.68	0.126	0.741	0.536	0.497	−0.037	−0.032

SEM = standard error of the mean.

Values with different superscripts in the same row are significantly different (P < 0.05).

3.3. Activity of digestive enzyme in hepatopancreas and intestine

Hepatopancreatic protease activity was significantly elevated in the 452.93 mg/kg group compared to the control (P = 0.043; Table 4). Hepatopancreatic amylase activity was significantly higher in all L-carnosine supplementation groups (59.28–821.16 mg/kg) versus the control (P < 0.001), with the peak value observed at 452.93 mg/kg.

Table 4.

Effects of dietary L-carnosine levels on the activities of digestive enzymes in hepatopancreas and intestines of red swamp crayfish.

Items	Dietary L-carnosine levels, mg/kg						SEM	P-value			Adjusted R2
	3.35	59.28	106.84	222.20	452.93	821.16		ANOVA	Linear	Quadratic	Linear	Quadratic
Hepatopancreas
Protease, U/g prot	139.19b	142.59ab	143.07ab	143.16ab	144.06a	141.12ab	0.514	0.043	0.762	0.015	−0.056	0.351
Amylase, U/mg prot	10.52b	14.29a	14.32a	14.37a	14.40a	14.19a	0.349	<0.001	0.096	0.013	0.111	0.364
Lipase, U/g prot	6.90	7.07	7.09	7.18	7.19	7.06	0.060	0.817	0.614	0.348	−0.045	0.015
Intestine
Protease, U/g prot	22.43c	22.44c	22.99c	23.22c	26.01a	24.73b	0.290	<0.001	<0.001	<0.001	0.520	0.731
Amylase, U/mg prot	4.29b	4.45b	6.14a	6.36a	6.39a	5.96a	0.219	<0.001	0.024	<0.001	0.236	0.672
Lipase, U/g prot	0.92	0.93	0.96	0.98	0.99	0.94	0.010	0.330	0.580	0.047	−0.042	0.247

prot = protein; SEM = standard error of the mean.

Values with different superscripts in the same row are significantly different (P < 0.05), n = 6.

Intestinal protease activity was significantly higher in the 452.93 and 821.16 mg/kg groups compared to the control (P < 0.001). Intestinal amylase activity was significantly elevated at L-carnosine levels ≥ 106.84 mg/kg (P < 0.001), also reaching its maximum at 452.93 mg/kg. Lipase activity in both the hepatopancreas and intestine remained unaffected across all treatment levels (P > 0.05).

Both hepatopancreatic and intestinal digestive enzyme activities showed significant quadratic responses to increasing dietary L-carnosine levels (P < 0.05). The enhancement occurred in a dose-dependent manner, with maximal activity consistently observed at 452.93 mg/kg.

3.4. Muscular activity of antioxidant enzyme

At dietary L-carnosine levels of 106.84 to 452.93 mg/kg, SOD activity was significantly higher compared to the control group (P < 0.001), with peak values observed at 452.93 mg/kg (Table 5). Catalase activity was significantly elevated in the 452.93 mg/kg group versus the control (P = 0.002) and reached its maximum at this level. The T-AOC was significantly higher at L-carnosine levels of 222.20 to 452.93 mg/kg compared to the control (P = 0.005), achieving its peak at 452.93 mg/kg. Conversely, MDA content in the muscle remained unaffected across all treatment levels (P = 0.988). The antioxidant parameters (SOD, CAT, and T-AOC) exhibited significant quadratic responses to increasing dietary L-carnosine levels (P < 0.001).

Table 5.

Effects of dietary L-carnosine levels on the antioxidant capacity, muscular textural properties and collagen contents in the muscle of red swamp crayfish.

Items	Dietary L-carnosine levels, mg/kg						SEM	P-value			Adjusted R2
	3.35	59.28	106.84	222.20	452.93	821.16		ANOVA	Linear	Quadratic	Linear	Quadratic
Antioxidant capacity
MDA, nmol/g prot	137.85	133.41	133.80	134.77	134.77	134.63	1.658	0.988	0.876	0.955	−0.061	−0.126
SOD, U/mg prot	2.37c	2.42bc	2.59ab	2.73a	2.74a	2.44bc	0.039	<0.001	0.632	<0.001	−0.047	0.799
CAT, U/mg prot	0.57b	0.56b	0.57b	0.64ab	0.75a	0.63ab	0.019	0.002	0.034	<0.001	0.204	0.609
T-AOC, nmol/g prot	32.05c	33.64bc	36.80abc	38.04ab	39.32a	34.64abc	0.725	0.005	0.349	<0.001	−0.004	0.628
Textural properties
Hardness, gf	354.83b	357.33b	377.83b	379.17b	417.67a	376.67b	4.141	<0.001	0.013	<0.001	0.143	0.592
Springiness	0.46	0.45	0.45	0.44	0.46	0.45	0.005	0.891	0.967	0.972	−0.029	−0.059
Chewiness, gf	56.42c	56.42c	63.60b	68.75b	77.98a	67.94b	1.348	<0.001	<0.001	<0.001	0.322	0.811
Resilience	0.23	0.23	0.23	0.22	0.22	0.23	0.004	0.992	0.842	0.955	−0.028	−0.058
Gumminess	134.24c	145.41bc	151.59b	150.83b	172.67a	147.27b	2.200	<0.001	0.043	<0.001	0.089	0.655
Hyp, g/kg
Alkaline-soluble Hyp	0.11c	0.12bc	0.13ab	0.13ab	0.14a	0.13ab	0.002	<0.001	0.008	<0.001	0.008	0.790
Alkaline-insoluble Hyp	1.07c	1.11bc	1.12bc	1.18abc	1.28a	1.23ab	0.021	0.004	0.002	<0.001	0.002	0.668
Total Hyp	1.18c	1.18c	1.25bc	1.31abc	1.43a	1.36ab	0.024	<0.001	0.001	<0.001	0.001	0.756

MDA = malondialdehyde; prot = protein; SOD = superoxide dismutase; CAT = catalase; T-AOC = total antioxidant capacity; Hyp = hydroxyproline; SEM = standard error of the mean.

Values with different superscripts in the same row are significantly different (P < 0.05), n = 6.

3.5. Muscular texture properties and collagen content

Hardness, chewiness, gumminess, alkali-soluble hydroxyproline content, alkali-insoluble hydroxyproline content, and total hydroxyproline content all exhibited linear and quadratic trends in response to dietary L-carnosine levels (P < 0.05; Table 5). Maximum hardness was achieved at 452.93 mg/kg, with hardness values demonstrating 17.71% elevation compared to the control group (P < 0.001). At L-carnosine levels of 106.84 to 821.16 mg/kg, the chewiness, gumminess, and alkali-soluble hydroxyproline content were all higher compared to the control group and achieved the maximum values at 452.93 mg/kg (P < 0.001). The alkali-insoluble hydroxyproline content and the total hydroxyproline content at L-carnosine levels of 452.93 to 821.16 mg/kg were both significantly higher compared to the control group (P < 0.05) and achieved peak values at 452.93 mg/kg. However, muscular springiness and resilience remained unaffected across all treatment levels (P > 0.05).

3.6. Histological analysis of muscle

Myofiber density and small-sized myofibers (<50 μm) percentage exhibited quadratic trends in response to dietary L-carnosine levels (P = 0.005; Fig. 2A and B). Histological quantification revealed optimal myofiber density at 452.93 mg/kg, where myofiber packing density increased 8.36% versus the control group (P = 0.011; Fig. 2C). At L-carnosine levels of 222.20 to 452.93 mg/kg, the percentage of myofibers with a diameter > 70 μm decreased 18.97% versus the control group (P = 0.012; Fig. 2B). The small-sized myofibers (<50 μm) percentage achieved peak value at 452.93 mg/kg and increased 43.17% versus the control group (P = 0.024). However, the myofibers between 50 and 70 μm diameter percentage remained unaffected across all treatment levels (P = 0.903).

Fig. 2.

The morphology of myofiber of red swamp crayfish after dietary L-carnosine treatment for 56 d. (A) Myofiber microstructure of cross-section (magnification 100×). (B) The density in the myofiber of red swamp crayfish. (C) The lengths of diameter in myofiber of red swamp crayfish. MF = myofiber. Different lowercase letters above the vertical bars represent significant differences (P < 0.05), n = 6. A, P-value of the variance analyzed by one-way ANOVA; L, P-value of linear trend analyzed by orthogonal polynomial contrasts; and Q, P-value of quadratic trend analyzed by orthogonal polynomial contrasts.

3.7. Hydrolyzed amino acid in muscle

The contents of total essential amino acids (ΣEAA), total non-essential amino acids (ΣNEAA), and total amino acids (ΣTAA) in the red swamp crayfish muscle all exhibited linear and quadratic trends with increasing levels of dietary L-carnosine (P < 0.05; Table 6). At L-carnosine levels of 452.93 to 821.16 mg/kg, the arginine, histidine, isoleucine, lysine, and ΣTAA contents were all higher versus the control group (P < 0.05) and achieved the maximum values at 452.93 mg/kg. The phenylalanine, valine, alanine, and ΣEAA contents at 452.93 mg/kg were all higher versus the control group (P < 0.05) and achieved the maximum values. At L-carnosine levels of 222.20 to 821.16 mg/kg, the contents of aspartic acid and ΣNEAA were higher versus the control group (P < 0.001) and achieved the maximum values at 452.93 mg/kg. The glycine content at L-carnosine levels of 59.28 to 821.16 mg/kg was higher versus the control group (P < 0.001). The proline content at L-carnosine levels of 59.28 to 452.93 mg/kg was higher versus the control group (P = 0.003). However, the contents of methionine, leucine, threonine, cysteine, glutamate, serine, and tyrosine in the muscle remained unaffected across all treatment levels (P > 0.05).

Table 6.

Effects of dietary L-carnosine levels on the amino acid composition in muscle of red swamp crayfish (g/kg, wet mass).

Items	Dietary L-carnosine levels, mg/kg						SEM	P-value			Adjusted R2
	3.35	59.28	106.84	222.20	452.93	821.16		ANOVA	Linear	Quadratic	Linear	Quadratic
Essential amino acids
Arginine	14.61b	15.21ab	15.30ab	15.68ab	16.29a	16.03a	0.161	0.006	0.002	<0.001	0.424	0.648
Histidine	4.51b	4.55b	4.61b	4.63b	5.06a	4.73ab	0.052	0.005	0.036	0.002	0.201	0.508
Isoleucine	7.89c	8.04bc	8.13bc	8.15bc	8.75a	8.46ab	0.080	0.002	0.003	<0.001	0.407	0.613
Leucine	12.01	12.19	12.22	12.26	13.10	12.69	0.158	0.392	0.078	0.100	0.130	0.166
Lysine	12.44b	13.16ab	13.28ab	13.39ab	14.12a	13.72a	0.151	0.009	0.009	0.001	0.318	0.568
Methionine	5.41	5.41	5.45	5.54	5.87	5.61	0.057	0.144	0.067	0.026	0.144	0.303
Phenylalanine	6.541b	6.60b	6.70ab	6.73ab	7.17a	6.94ab	0.067	0.025	0.008	0.003	0.320	0.484
Threonine	6.38	6.45	6.46	6.91	7.03	6.80	0.095	0.205	0.077	0.026	0.131	0.303
Valine	6.84b	6.91b	7.02ab	7.03ab	7.54a	7.28ab	0.074	0.025	0.009	0.003	0.311	0.471
Non-essential amino acids
Alanine	8.49b	8.61b	8.67b	8.98ab	9.34a	9.01ab	0.084	0.007	0.010	<0.001	0.307	0.637
Aspartic acid	15.06c	15.45c	15.79bc	16.58ab	16.81a	16.49ab	0.169	<0.001	0.002	<0.001	0.417	0.800
Cysteine	6.23	5.88	6.26	6.50	6.90	6.44	0.073	0.106	0.114	0.034	0.095	0.278
Glycine	7.53b	8.05a	8.12a	8.33a	8.34a	8.29a	0.267	<0.001	0.012	<0.001	0.294	0.623
Glutamate	20.12	20.81	20.96	21.82	23.04	22.23	0.038	0.001	0.002	<0.001	0.428	0.741
Proline	5.42b	5.71a	5.73a	5.78a	5.85a	5.65ab	0.093	0.003	0.346	0.002	−0.003	0.508
Serine	6.54	6.43	6.66	6.99	7.12	6.84	0.092	0.230	0.124	0.038	0.088	0.268
Tyrosine	7.74	7.66	7.78	7.92	8.37	8.05	0.107	0.245	0.073	0.051	0.137	0.237
ΣEAA	76.63b	78.52b	79.16ab	80.32ab	84.94a	82.26ab	0.794	0.011	0.006	0.001	0.350	0.586
ΣNEAA	77.26c	78.54bc	79.90bc	82.76ab	85.96a	83.17ab	0.822	0.001	0.004	<0.001	0.377	0.738
ΣTAA	153.90c	157.06bc	159.07bc	163.08abc	170.90a	165.43ab	1.594	0.004	0.004	<0.001	0.375	0.682

ΣEAA = total essential amino acids; ΣNEAA = total non-essential amino acids; and ΣTAA = total amino acids; SEM = standard error of the mean.

Values with different superscripts in the same row are significantly different (P < 0.05), n = 6.

3.8. Muscle gene expression

The 452.93 mg/kg level enhanced the protein synthesis signaling pathway. At 452.93 mg/kg, the mRNA expression levels of igf-1, pi3k, akt, tor, and s6k1 were upregulated versus the control group (P < 0.05; Fig. 3). Conversely, the mRNA expression levels of 4ebp1 and eif2a were downregulated versus the control group (P < 0.001). At 452.93 mg/kg, the mRNA expression levels of genes related to the ubiquitin-proteasome and autophagy–lysosome pathways, including ub, psma2, psmc1, murf1, atg5, atg16l1, beclin1, and atg12 were all higher versus the control group (P < 0.05; Fig. 4). Collagen biosynthesis demonstrated earlier transcriptional activation, with TGF-β/Smad signaling components (TGF-β1, Smad3/4/6, and Col1α1) showing peak at 222.20 mg/kg versus the control group (P < 0.05; Fig. 5). The mRNA expression levels of genes related to muscle development, including mef2a and mef2b, were all higher at L-carnosine levels of 452.93 to 821.16 mg/kg versus the control group (P < 0.001; Fig. 6). The mRNA expression level of mstn was lower at L-carnosine levels of 59.28 to 821.16 mg/kg versus the control group (P < 0.001). At L-carnosine levels of 59.28 to 821.16 mg/kg, the mRNA expression level of smyhc15 was higher versus the control group (P < 0.001). At L-carnosine levels of 222.20 to 452.93 mg/kg, the mRNA expression level of smyhc1 was lower versus the control group (P < 0.001).

Fig. 3.

Effects of dietary L-carnosine levels on the protein synthesis-related genes of red swamp crayfish. Different lowercase letters above the vertical bars represent significant differences (P < 0.05), n = 6. A, P-value of the variance analyzed by one-way ANOVA; L, P-value of linear trend analyzed by orthogonal polynomial contrasts; and Q, P-value of quadratic trend analyzed by orthogonal polynomial contrasts.

Fig. 4.

Effects of dietary L-carnosine levels on the protein degradation-related genes of red swamp crayfish. Different lowercase letters above the vertical bars represent significant differences (P < 0.05), n = 6. A, P-value of the variance analyzed by one-way ANOVA; L, P-value of linear trend analyzed by orthogonal polynomial contrasts; and Q, P-value of quadratic trend analyzed by orthogonal polynomial contrasts.

Fig. 5.

Effects of dietary L-carnosine levels on the regulatory factors of Collagen synthesis and metabolism of red swamp crayfish. Different lowercase letters above the vertical bars represent significant differences (P < 0.05), n = 6. A, P-value of the variance analyzed by one-way ANOVA; L, P-value of linear trend analyzed by orthogonal polynomial contrasts; and Q, P-value of quadratic trend analyzed by orthogonal polynomial contrasts.

Fig. 6.

Effects of dietary L-carnosine levels on the muscle development and muscle fiber transformation related genes of red swamp crayfish. Different lowercase letters above the vertical bars represent significant differences (P < 0.05), n = 6. A, P-value of the variance analyzed by one-way ANOVA; L, P-value of linear trend analyzed by orthogonal polynomial contrasts; and Q, P-value of quadratic trend analyzed by orthogonal polynomial contrasts.

4. Discussion

4.1. Dietary L-carnosine improved the feed utilization and growth

The growth-promoting benefits of dietary L-carnosine observed in red swamp crayfish align with previous findings in E. coioides (Li et al., 2024), juvenile turbot (Wang et al., 2017), broiler chickens (Cong et al., 2017), and finishing pigs (Bao et al., 2015). Notably, this nutritional strategy exhibited species specificity, as evidenced by null effects in rainbow trout (Snyder et al., 2012) and finishing pigs (Ma et al., 2010). The observed efficacy in red swamp crayfish likely stems from its unique crustacean physiology characterized by discontinuous growth mediated through periodic molting cycles (Vijayan et al., 2003). In addition, dietary L-carnosine increased the activity of protease and amylase in the hepatopancreas and intestine of the red swamp crayfish. Enhanced digestive enzyme activity facilitated the absorption and utilization of dietary nutrients, providing energy for the periodic molting process (Yao et al., 2024). Although the two principal digestive organs exhibited slightly different response thresholds, dietary L-carnosine collectively improved their enzymatic function, enhancing the overall capacity of the digestive system and facilitating more efficient nutrient breakdown and absorption.

The improved nutrient utilization was ultimately reflected in the body composition of the crayfish. It is noteworthy that dietary L-carnosine significantly elevated the crude protein content in both the whole body and the abdominal muscle, while the moisture, lipid, and ash contents remained stable. This pattern demonstrates that enhanced growth performance was primarily driven by a specific stimulation of protein deposition rather than a general increase in all body components. The rise in whole-body protein directly corroborates the observed improvements in PDR and PER, confirming that dietary protein was more efficiently utilized for tissue growth. Furthermore, the synchronous increase in crude protein at both the whole-body and abdominal muscle levels reveals a coordinated anabolic response. Crucially, the magnitude of increase in muscle crude protein was more pronounced than that in the whole body. This discrepancy indicates that the protein deposited under dietary L-carnosine was preferentially partitioned towards the abdominal muscle, the primary edible portion. This targeted deposition not only directly explains the significant rise in the muscle percentage but also underscores that the improvement in overall growth represents a substantial enhancement in the yield of high-value tissue, rather than merely an increase in total mass. These findings align with observations in orange-spotted grouper (Li et al., 2024).

Concurrently, the nutritional quality of the muscle was substantially improved, as evidenced by the significantly increased contents of ΣTAA, ΣEAA, and several individual amino acids—most notably arginine, histidine, glycine, and proline at the optimal dietary L-carnosine level (452.93 mg/kg). The rise in these amino acids is critical, as they serve not only as the fundamental building blocks for protein deposition but also play specific metabolic roles. The marked increase in arginine, a limiting amino acid in crustacean metabolism and a precursor for phosphoarginine in the phosphagen system, may directly support the high energy demands associated with molting and post-molt muscle regeneration (Li et al., 2022). The elevated levels of glycine and proline, key components of collagen, align with the observed improvements in muscle texture and collagen content (Tian et al., 2023). In addition, it was recently established in this laboratory that histidine stimulates molting hormone secretion in red swamp crayfish, promoting the molting process and thereby enhancing their growth performance (Qi et al., 2019; Yao et al., 2024). Collectively, these findings indicate that L-carnosine promotes the red swamp crayfish growth performance by improving nutrient utilization.

4.2. Dietary L-carnosine improved the muscle antioxidant capacity

Dietary L-carnosine improved the muscle antioxidant capacity of red swamp crayfish. Excessive dietary plant protein induces excessive production of free radicals, especially reactive oxygen species (ROS), which leads to the oxidation of lipids and proteins in muscle (Liu et al., 2024). Protein oxidation leads to a decrease in muscle tenderness, water-holding capacity, and color (Cong et al., 2017). Lipid oxidation negatively affects muscle texture, flavor, color, nutritional value, and safety, and also reduces muscle water-holding capacity (Cong et al., 2017). The antioxidant capacity-enhancing effects of dietary L-carnosine observed in red swamp crayfish corroborate existing vertebrate studies, including those of the orange-spotted grouper (Li et al., 2024), broiler chickens (Cong et al., 2017), and finishing pigs (Ma et al., 2010). This cross-species conservation suggests a fundamental mechanism whereby L-carnosine exerts the dual capacity of L-carnosine for metal ion chelation and free radical scavenging (Cong et al., 2017). Excessive oxidation of lipids and protein can reduce the quality of meat by altering their composition of lipids and protein (Liu et al., 2024). Previous experiments from this laboratory confirmed that increasing muscle antioxidant capacity improves muscle quality by increasing tenderness and flavor (Li et al., 2022; Tian et al., 2022).

4.3. Dietary L-carnosine improved muscle hardness

Dietary L-carnosine (452.93 mg/kg) significantly enhanced muscle hardness and chewiness in red swamp crayfish, reinforcing its role as a primary textural determinant in aquatic products. In addition to red swamp crayfish, these improvements mirror textural enhancements documented in L-carnosine-supplemented orange-spotted grouper (Li et al., 2024), broiler chickens (Cong et al., 2017), and finishing pigs (Bao et al., 2015). Notably, sarcomeric reorganization suggests enhanced myofibril alignment, potentially explaining the chewiness elevation in grass carp (Ctenopharyngodon idellus) (Tian et al., 2022) and Pacific white shrimp (Litopenaeus vannamei) (Li et al., 2022).

In addition, as the principal constituent of muscle connective tissue, collagen critically also determines textural hardness through its crosslinking maturity (Liu et al., 2005; Tian et al., 2022). This structural paradigm has been quantitatively validated in aquacultured Atlantic salmon (Salmo salar L.), where myofibrillar collagen content exhibited a significant positive correlation with tissue hardness indices (Aidos et al., 1999). It was revealed by nutritional intervention trials in this laboratory that dietary L-carnosine effectively increased the content of alkali-insoluble hydroxyproline and alkali-soluble hydroxyproline in the red swamp crayfish muscle. These biochemical alterations indicate that the dipeptide enhances muscle hardness through elevated mature collagen fractions, probably by stabilization of pyridinoline cross-linked networks.

The TGF-β1 signaling pathway is a key regulator of myofibrillar collagen biosynthesis, primarily through the transcriptional activation of extracellular matrix-related genes (Yu et al., 2019). In the present study, the transcription of key components within the TGF-β1/Smads pathway was upregulated in response to dietary L-carnosine. Type I collagen, a heterotrimer composed of two α1 and one α2 subunits, is known to be transcriptionally coregulated with TGF-β1 and its downstream effectors Smad2/4 (Yu et al., 2019). Mechanistically, activated Smad3 can promote the expression of tissue inhibitors of TIMPs while MMP activity (Johnston et al., 2006), and TGF-β1 itself also inhibits MMP activity (Liu et al., 2005). The present data revealed that dietary L-carnosine promoted TGF-β1 and Smad3 upregulation. Taken together, these transcriptional changes, along with established literature, suggest that L-carnosine could attenuate collagen degradation by enhancing the TGF-β1/Smads signaling cascade. In addition, parallel regulatory mechanisms may involve pathways such as PI3K/Akt/S6K1, through which factors like IGF-1 can modulate LARP6 expression to stabilize collagen mRNA and ensure translational fidelity (Parsons et al., 2011). Therefore, the present study proposes that dietary L-carnosine may influence type I collagen metabolism in crayfish muscle through a combined action: potentially reinforcing the TGF-β1/Smads-mediated anti-degradative pathway and possibly cooperating with translation–regulatory axes such as PI3K/Akt/S6K1. The full names of the abbreviations of proteins can be found in Table S3.

4.4. Dietary L-carnosine improved muscle development

Muscle development is dynamically regulated by nutritional status (Jagot et al., 2018). The present findings suggest that dietary L-carnosine may promote muscle development in red swamp crayfish through a multi-faceted modulation of gene expression related to protein metabolism, potentially involving coordinated upregulation of anabolic processes and downregulation of catabolic pathways.

Dietary L-carnosine directly influenced the expression of key myogenic regulators. It significantly upregulated the transcript levels of myocyte enhancer factors (mef2a and mef2b), which are central to myogenesis (Taylor and Hughes, 2017), while concurrently downregulating the expression of MSTN, a potent negative regulator of muscle growth (Baczek et al., 2020; Dong et al., 2022). This shift in the transcriptional profile is consistent with the establishment of a molecular environment favorable for myofiber hypertrophy and proliferation, which aligns with the observed increase in abdominal muscle percentage.

Muscle growth is influenced not only by muscle cell proliferation but also by protein deposition (Wilschut et al., 2010). It was previously found in this laboratory that histidine-enriched diets enhance nitrogen retention through improved PER and PDR (Yao et al., 2024). Dietary L-carnosine supplementation has similar effects in rainbow trout and red swamp crayfish (Snyder et al., 2012). This nutritional strategy indicated that more abundant the histidine pool, the more favorable the protein deposition. Protein deposition depends on amino acid deposition, and the increase in amino acid content is the basis for the increase in protein content (Li et al., 2022). Results from this study reveal that L-carnosine dietary intervention enhanced the muscle crude protein, ΣTAA, and ΣEAA content, paralleling observations in orange-spotted grouper (Li et al., 2024).

To explore potential mechanisms underlying enhanced protein deposition, pathways governing protein synthesis were examined. The mTORC1 pathway is an essential regulator of protein anabolism (Liu et al., 2022; Goodman et al., 2011). Transcriptional profiling in this work showed changes in the expression of several mTORC1 signaling components. These consistent results are demonstrated in pigs (Liu et al., 2022), fine flounder (Paralichthys olivaceus) (Valenzuela et al., 2015), zebrafish (Brachydanio rerio var) (Palstra et al., 2014), and gilthead sea bream (Sparus aurata) (Vélez et al., 2016). Based on these transcriptional changes and the established role of this pathway, it is plausible that dietary L-carnosine might contribute to enhanced protein deposition in part by modulating the mTORC1 signaling network. Beyond mTORC1, the eIF2B-eIF2 pathway critically regulates translation initiation by mediating GTP-dependent peptide chain assembly (Goodman et al., 2011). Furthermore, dietary L-carnosine downregulated eif2α transcription. As the eIF2B-eIF2 pathway critically regulates translation initiation, this observation suggests a possible role for dietary L-carnosine in promoting protein translation initiation through this mechanism.

This study showed that dietary L-carnosine significantly downregulated the mRNA expression of core regulatory components in both proteolytic pathways. Both systems collectively mediate cellular proteolysis-the ubiquitin-proteasome pathway eliminates misfolded polypeptides through targeted degradation, maintaining proteostasis, while autophagic mechanisms clear damaged organelles via lysosomal processing (Cao and Klionsky, 2007; Nemova et al., 2016; Saera-Vila et al., 2016; Todgham et al., 2017; Witt et al., 2005). Comparative physiological studies reveal evolutionary conservation of these mechanisms: Antarctic fish (Pachycara brachycephalum) exhibit proteasome-mediated muscle remodeling under environmental stress (Windisch et al., 2014), whereas turbot (Scophthalmus maximus L.) displays autophagy-dependent protein catabolism during metabolic adaptation (Xu et al., 2015). These findings collectively imply that dietary L-carnosine may attenuate proteolytic processes by coordinately downregulating both proteolytic pathways.

In conclusion, the transcriptional evidence presented in this study supports a model in which dietary L-carnosine may establish a positive protein balance in crayfish muscle by potentially stimulating synthesis-related pathways and inhibiting degradation-related pathways at the transcriptional level, thereby providing a mechanistic explanation for the observed enhancements in muscle growth and development.

4.5. Determination of the optimal dietary L-carnosine level

The dose–response relationships observed in this study indicate that dietary L-carnosine influences red swamp crayfish in a quadratic manner, with a defined optimal range of 425 to 500 mg/kg as determined by quadratic regression modeling. A notable finding was the consistent decline in key performance variables at the supra-optimal level of 821.16 mg/kg. This pattern follows a well-established principle in nutritional physiology, where many functional nutrients exhibit diminished benefits or even adverse effects beyond an optimal dosage (NRC, 2011).

Although the precise mechanisms underlying this decline were not directly investigated in this study, several physiologically plausible explanations can be considered based on general nutrient-response principles. The reduction in efficacy at 821.16 mg/kg may reflect a saturation of the beneficial pathways activated at lower doses. One potential mechanism is nutrient imbalance: excessively high L-carnosine may compete with structurally related metabolites such as histidine and alanine for shared membrane transporters or metabolic pathways, thereby disrupting their homeostasis and utilization (Yao et al., 2024). Additionally, L-carnosine is known to function as a pH-buffer and antioxidant at physiological levels; however, at very high concentrations, it might paradoxically perturb cellular redox balance or ionic equilibrium, leading to metabolic stress (Diniz et al., 2024).

Anabolic signaling pathways such as mTOR, which often mediate nutrient-sensitive growth responses, typically exhibit saturable activation; beyond a certain threshold, further stimulation may not enhance anabolism and could even induce feedback inhibition or desensitization (Liu et al., 2022). It is thus plausible that the decline in growth performance at the highest dose reflects such a saturation or over-stimulation of key regulatory networks.

In summary, while the exact mechanisms require further validation, the decline in efficacy at the supra-optimal dose underscores the importance of dose optimization in nutritional interventions. These observations reinforce that 458.06 to 500.00 mg/kg represents the most effective and economical dietary L-carnosine level for red swamp crayfish under the present experimental conditions.

5. Conclusion

Dietary L-carnosine at the optimal level (458.06–500.00 mg/kg, determined by quadratic regression modeling) significantly improved growth performance, protein utilization, feed efficiency and muscle percentage in red swamp crayfish. It also increased muscle hardness, which corresponded with elevated myofiber density and upregulation of genes in the TGF-β1/Smads signaling pathway—a pathway involved in collagen synthesis in muscle tissue. In addition, L-carnosine supplementation improved muscle percentage and nutritional quality, accompanied by evidence of myofiber hypertrophy and proliferation, as reflected by the transcriptional upregulation of key growth-related factors. The mRNA expression patterns further suggested that dietary L-carnosine may support muscle protein deposition through modulation of the mTORC1 and eIF2B-eIF2 pathways.

Credit Author Statement

Xinyuan Li: Writing – original draft, Data curation. Yangyang Liu: Methodology. Shouqi Xie: Funding acquisition. Jianmin Zhang: Methodology, Conceptualization. Weihua Gao: Funding acquisition. Ming Jiang: Conceptualization. Lixue Dong: Methodology. Di Peng: Methodology. Ke Cheng: Methodology. Feng Huang: Conceptualization. Juan Tian: Writing – review & editing, Funding acquisition, Conceptualization.

Declaration of generative AI in scientific writing

During the preparation of this manuscript, we used Chatgpt (OpenAI) only to improve the language in several parts of the manuscript. After using this tool, we reviewed and edited the content as needed and take full responsibility for the final content of the published article.

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. Ming Jiang is an Editorial Board Member for Animal Nutrition and was not involved the editorial review or the decision to publish this article.

Appendix A. Supplementary data

The following is the Supplementary data to this article.