High Oleic Acid Diet Promotes Growth and Muscle Metabolic Remodeling in Eriocheir sinensis: Multi-Omics Insight into Lipid Deposition and Nutrient Quality

TL;DR

A high oleic acid diet improves growth and muscle quality in Chinese mitten crabs by altering lipid metabolism and amino acid profiles.

Contribution

This study reveals how high oleic acid diets reprogram muscle metabolism in crabs through multi-omics analysis.

Findings

HOA diet increased growth rate and protein efficiency without affecting survival or organ indices.

HOA enhanced antioxidant capacity and altered muscle lipid metabolism without increasing oleic acid content.

HOA shifted amino acid profiles and triggered glycerophospholipid remodeling in crab muscle.

Abstract

Dietary lipid sources critically influence growth, health, and muscle quality in Chinese mitten crab (Eriocheir sinensis), yet how high oleic acid diet (HOA) regulates intramuscular nutrient deposition remains unclear. Here, a 10-week feeding trial compared isonitrogenous and isoenergetic diets, in which soybean oil was replaced with high-oleic peanut oil. HOA significantly improved weight gain, specific growth rate, and protein efficiency ratio, without affecting survival, hepatosomatic index (HSI), or gonadosomatic index (GSI). HOA enhanced antioxidant capacity by increasing catalase activity and reducing malondialdehyde, while key non-specific immune enzymes were unchanged. In muscle, HOA did not increase intramuscular oleic acid (OA) content but reduced linoleic acid and upregulated genes involved in fatty acid transport and β-oxidation. HOA also shifted free amino acids (higher glutamate and lysine; lower proline) without significant transcriptional upregulation of the mechanistic target of rapamycin (mTOR) pathway or changing total protein. Multi-omics analyses indicated altered nucleotide/purine pathways and pronounced glycerophospholipid remodeling, identifying discriminatory lipid species. Overall, oleic-acid-rich lipids promote growth and antioxidant defense while reprogramming muscle lipid metabolism, supporting their targeted use to optimize crab muscle quality.

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1. Introduction

The Chinese mitten crab (Eriocheir sinensis) is one of the most economically important freshwater crustaceans and is highly popular among consumers in China and Southeast Asia due to its distinctive flavor and rich nutritional profile, characterized by high levels of fatty acids and amino acids [1,2]. As one of the primary edible parts of the crab, the muscle tissue largely determines its sensory and nutritional quality, which in turn directly influences product acceptability and commercial value [3]. Nutrient regulation thus represents a key strategy for improving muscle quality. Accordingly, a major focus in enhancing E. sinensis quality lies in clarifying how nutrients are accumulated in muscle and how they contribute to flavor development.

Dietary lipids are crucial for aquatic animals, serving not only as a digestible energy source and essential fatty acids, but also supporting membrane structure and immune function [4,5,6]. During the fattening period, E. sinensis exhibits high demands for energy and lipids to support rapid growth and nutrient storage [7]. While high-fat diets can improve growth and feed efficiency through a protein-sparing effect [8], excessive lipid intake can lead to metabolic disturbances, such as impaired antioxidant capacity and abnormal lipid deposition, ultimately compromising growth and health [9,10]. Therefore, the rational selection of lipid sources is critical.

Oleic acid (OA; 18:1n-9) is a monounsaturated fatty acid widely present in plant and animal oils and represents a major constituent of monounsaturated fatty acids. As an efficient substrate for β-oxidation, OA can be readily catabolized and utilized, thereby providing energy to support growth and nutrient deposition [11,12,13]. In Chinese mitten crab larvae, compared with perilla oil rich in α-linolenic acid, feeding olive oil rich in OA promotes glucose and lipid catabolism, enhancing energy supply and ultimately benefiting growth [8]. Similar growth-promoting effects have also been reported in hybrid grouper [6]. Moreover, OA can modify the fatty acid profile and increase the proportion of OA in muscle; it can also reduce the levels of mTOR-related proteins [14]. Both in vivo and in vitro studies further suggest an association between OA and lipid deposition [15,16]. Beyond its metabolic roles, OA also shows potential benefits in antioxidative capacity and immune modulation. In species such as European seabass, OA supplementation helps alleviate oxidative stress induced by high-fat diets [10] and can enhance immune responses and disease resistance, thus being considered a potential immunostimulant [6,17,18]. However, systematic studies on the physiological roles of OA in the Chinese mitten crab during the fattening stage and its mechanisms regulating muscle nutrient deposition are lacking.

To address the knowledge gap, we examined the effects of high oleic acid diet in nutrient deposition in E. sinensis. Growth and physiological status were evaluated using growth indices, antioxidant and non-specific immune parameters, and muscle fatty acid and free amino acid profiles. Mechanisms were investigated by integrating muscle lipidomics and metabolomics with qRT-PCR of genes related to fatty acid transport, mitochondrial β-oxidation, amino acid metabolism, and protein synthesis signaling. The HOA improved growth and protein utilization and enhanced antioxidant capacity, accompanied by upregulated fatty acid transport and β-oxidation and broad remodeling of muscle lipids and metabolites.

2. Results

2.1. Effects of the High Oleic Acid Diet on Growth and Development of E. sinensis

The HOA significantly enhanced the growth performance of E. sinensis, as evidenced by increased weight gain, specific growth rate, and protein efficiency ratio compared to the standard diet group (Figure 1B–D). Notably, this growth promotion occurred without compromising survival (Figure 1A) or significantly altering the hepatosomatic and gonadosomatic indices (Figure 1E,F). These results collectively indicate that the high oleic acid diet (HOA) effectively improves growth and protein utilization efficiency in E. sinensis, while maintaining normal survival and organ development.

2.2. Effects of the HOA on Antioxidant Capacity and Non-Specific Immunity in E. sinensis

The HOA did not significantly affect the activities of total antioxidant capacity (T-AOC), total superoxide dismutase (T-SOD), or glutathione (GSH) in the hemolymph (Figure 2A,B,D). In contrast, it significantly elevated catalase (CAT) activity and markedly reduced malondialdehyde (MDA) content (Figure 2C,E). Similarly, no significant differences were observed in the activities of aspartate aminotransferase (GOT), acid phosphatase (ACP), and alkaline phosphatase (AKP) between the two groups (Figure 2F–H). These findings suggest that HOA enhances the antioxidant defense system in E. sinensis, potentially protecting tissues from oxidative damage without significantly influencing non-specific immunity.

2.3. The HOA Enhances Fatty Acid Transport and Catabolism in the Muscle of E. sinensis

To further evaluate the effects of HOA on fatty acid metabolism in E. sinensis, we first measured the concentrations of triglycerides (TG), total cholesterol (TC), and glucose in the hemolymph. The HOA did not significantly alter TG and TC levels compared with the standard diet group (Figure 3A–C). Subsequently, we analyzed the fatty acid composition of the muscle (Table 1; Figure 3D,E). Total monounsaturated fatty acids (MUFA), total saturated fatty acids (SFA), and total polyunsaturated fatty acids (PUFA) did not differ significantly between the two groups (Figure 3D). Similarly, oleic acid (C18:1) content remained unchanged (Figure 3E), whereas linoleic acid (C18:2) content was significantly decreased (p = 0.025; Figure 3E). In addition, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) levels did not differ significantly between groups (Figure 3E). To explore potential alterations in fatty acid metabolism, we further examined the expression of fatty acid–related genes (Figure 3F). Genes involved in fatty acid synthesis, such as sterol regulatory element-binding protein 1 (srebp1) and peroxisome proliferator-activated receptor gamma (ppar-γ), were not significantly affected. In contrast, genes associated with fatty acid transport and catabolism, including fatty acid transport protein 4 (fatp4), fatty acid-binding protein 9 (fabp9), lipoprotein lipase (lpl), glycerol-3-phosphate acyltransferase 3 (gpat3), carnitine palmitoyltransferase 1A (cpt1a), and carnitine palmitoyltransferase 1B (cpt1b), were significantly upregulated. Taken together, these findings suggest that HOA enhances fatty acid transport and catabolism in the muscle of E. sinensis.

2.4. Effects of HOA on Muscle Free Amino Acid Composition and Protein Synthesis in E. sinensis

To further elucidate the impact of HOA on the muscle amino acid profile of the E. sinensis, we measured changes in free amino acid levels and examined the expression of amino acid metabolism and protein synthesis related genes. The results indicated that free lysine and glutamic acid concentrations in the muscle were significantly elevated (p < 0.05; Table 2), whereas proline content was markedly decreased (p = 0.029; Table 2).

In contrast, the contents of free essential amino acids (free-EAA), free non-essential amino acids (free-NEAA), and free flavor amino acids (free-FAA) showed no significant differences compared with the control group (p > 0.05; Figure 4A). Regarding amino acid metabolism, the transcriptional level of glutamine synthetase 2(gs2), a gene associated with catabolism, was upregulated, while that of glutamate dehydrogenase (gdh) showed no significant change. Similarly, the expression of two genes involved in amino acid anabolism, elongation factor 1-alpha (eef1α) and eukaryotic initiation factor 2 (eif2), remained unchanged (p > 0.05; Figure 4B). No significant differences were observed in total protein (TP) content (p > 0.05; Figure 4C). The expression of key components within the mammalian target of rapamycin (mTOR) signaling pathway, including mtor itself, ribosomal protein S6 kinase 1 (s6k1), ribosomal protein S6 (s6), eukaryotic translation initiation factor 4E-binding protein 1 (4ebp1), and eukaryotic translation initiation factor 4E (eif4e), was also unaffected (p > 0.05; Figure 4D). Collectively, these results indicate that although HOA altered the free amino acid profile, it did not significantly affect overall protein synthesis in the muscle of E. sinensis.

2.5. Untargeted Metabolomics Analysis of HOA-Induced Metabolic Reprogramming in the Muscle of E. sinensis

Since intramuscular metabolism influences nutrient deposition and thereby modulates the nutritional value and sensory characteristics of muscle tissues, we applied untargeted metabolomics to comprehensively profile metabolite landscapes induced by HOA in the muscle of the E. sinensis. Principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) were employed to distinguish and classify the samples, respectively. PCA revealed no obvious separation between crabs fed HOA and those fed control diet (Figure 5A,D), indicating that HOA-induced metabolic variations were overshadowed by broader unsupervised variance within the dataset. However, clear differentiation was achieved with PLS-DA (Figure 5B,E), confirming that the HOA significantly influenced the metabolic profile of crab muscle. Subsequent multivariate analysis via PLS-DA identified 59 differentially expressed metabolites (DEMs) (VIP > 1, p < 0.05) detected in both positive and negative ionization modes. Compared with the standard diet group, the HOA group exhibited upregulation of 12 metabolites in each ionization mode, while 21 metabolites in positive mode and 14 in negative mode were downregulated (Figure 5C,F).

A total of 218 metabolites were identified and categorized into nine distinct classes (Figure 6A). These DEMs were then subjected to hierarchical clustering heatmap visualization and VIP value calculation (Figure 6B). Furthermore, KEGG enrichment analysis and KEGG topology analysis were conducted to elucidate the biological implications of these DEMs. The results highlighted enrichment of DEMs in pathways related to flavor substances and lipid metabolism, including nucleotide metabolism and biosynthesis of unsaturated fatty acids (Figure 6C,D). Using interaction networks, we visualized the relationships between these pathways and DEMs. A total of 7 DEMs were enriched across 5 pathways, with significant enrichment of guanosine, N6-(1,2-dicarboxyethyl)-AMP, adenylosuccinate, docosahexaenoic acid, linoleic acid, kanamycin, gentamicin C2, and inosine in pathways such as purine metabolism, nucleotide metabolism, biosynthesis of unsaturated fatty acids, linoleic acid metabolism, and neomycin/kanamycin/gentamicin biosynthesis (Figure 6E).

2.6. Targeted Lipidomics Reveals HOA-Induced Remodeling of Muscle Lipid Profiles in E. sinensis

To investigate the effects of HOA on lipid profiles in the muscle of E. sinensis, we conducted targeted lipidomics analysis of muscle tissues from HOA and control groups. PCA and PLS-DA revealed clear separation between the HOA and control groups, with tight clustering within each group (Figure 7A,B,D,E), suggesting distinct lipid profile variations induced by dietary treatment. A volcano plot showed that, compared with the control group, the HOA upregulated 95 lipids in positive mode and 54 in negative mode, while 27 positive-mode lipids and 51 negative-mode lipids were downregulated (Figure 7C,F).

A total of 227 lipids were identified and classified into four categories: glycerophospholipids (GP, 171 types), sphingolipids (SP, 30 types), glycerolipids (GL, 24 types), and fatty acids (FA, 2 types) (Figure 8A). Glycerophospholipids were the most abundant and predominant subclass among the identified lipids. No significant differences in overall lipid content or levels of MUFA were observed between groups (p > 0.05; Figure 8B,D). Based on VIP > 2 and p < 0.05 criteria, 30 differentially expressed lipids (DLMs) were selected (Figure 8C). ROC analysis was further performed on these DLMs to identify potential key lipids. The analysis revealed diacylglycerol (DG, 16:1e/8:0), phosphatidylethanolamine (PE, 18:0p/20:3), lysophosphatidylcholine (LPC, 20:3), phosphatidylcholine (PC, 16:1/19:0), and triacylglycerol (TG, 18:3/18:3/18:3) as critical metabolites with equivalent diagnostic potential (AUC = 1) (Figure 8E). KEGG pathway enrichment analysis based on differential abundance scores revealed that several metabolic pathways were affected, among which glycerophospholipid metabolism was significantly enriched (Figure 8F). KEGG topology analysis of these DLMs indicated significant enrichment in pathways related to glycerophospholipid metabolism, linoleic acid metabolism, and ether lipid metabolism (Figure 8G).

3. Discussion

Following the pubertal molt of the E. sinensis, multiple organ systems undergo rapid development, ultimately culminating in sexual maturation [19]. During this critical fattening period, the provision of dietary lipids is essential for the growth, development, and nutritional quality of crabs [20]. In the present study, HOA significantly improved growth performance, including WG, SGR, and PER, compared to the control group. These results suggest that HOA during maturation may improve both the productivity and profitability of crab aquaculture. The growth-promoting effect of HOA is consistent with findings in European sea bass (Dicentrarchus labrax), in which OA supplementation also improved feed conversion and protein utilization [21], likely attributable, at least in part, to OA serving as an efficient energy source. Additionally, HOA in E. sinensis did not significantly affect the hepatosomatic index (HSI) or gonadosomatic index (GSI). Therefore, we hypothesize that HOA may primarily influence nutrient deposition in the muscle tissues of E. sinensis, thereby potentially improving muscle quality.

The health status of the organism is crucial for the growth and development of E. sinensis, and the high demand for nutrients during its developmental stages makes it prone to oxidative stress [22]. Previous studies have demonstrated that OA can alleviate oxidative stress [23]. Therefore, we investigated the effect of HOA on crab health by assessing its impact on the antioxidant system. Oxidative stress can generate excessive reactive oxygen species (ROS), leading to lipid peroxidation. Aquatic animals counteract ROS-induced damage through antioxidant defense systems, with enzymes like T-SOD and GSH-Px playing key roles [24]. T-AOC reflects overall antioxidant status, while MDA serves as a biomarker of oxidative stress [25,26]. In this study, HOA had no significant effect on serum T-AOC, but it increased CAT activity and decreased MDA levels. Elevated CAT activity likely reduces hydroxyl radical generation, potentially mitigating lipid peroxidation as reflected by lower MDA levels [27,28].

Most crustacean species, including E. sinensis, lack adaptive immunity and mainly depend on innate immunity to resist pathogens [27]. AKP and ACP in the hemolymph form part of the first barrier of nonspecific immunity in crabs [29]. In this study, AKP and ACP activities remained unchanged across dietary treatments, suggesting that HOA health effects may be more closely associated with antioxidant regulation than with changes in these innate immune markers.

Nutrient deposition in muscle tissue directly influences quality traits [3]. Previous studies have shown that increased dietary OA promotes its accumulation in tissues and induces fat deposition [30,31]. In the present study, despite OA supplementation, neither muscle OA content nor TG levels increased significantly. This indicates that exogenous OA may have been directed toward metabolic pathways other than storage. To explore this possibility, we analyzed the expression of key genes involved in fatty acid metabolism in muscle tissue. The mRNA levels of srebp-1, a transcription factor regulating de novo fatty acid synthesis, and ppar-γ, which promotes adipocyte differentiation [32], remained unchanged, which may partially explain the absence of TG accumulation. In contrast, genes involved in fatty acid catabolism were upregulated, including lpl and fatp4 (uptake) [33], fabp9 (transport), and cpt1a and cpt1b (β-oxidation) [34,35]. These results suggest that dietary HOA may enhance fatty acid uptake and β-oxidation in muscle, favoring fatty acid use for energy to support growth rather than storage. Furthermore, HOA significantly reduced the linoleic acid (C18:2) content in muscle. Because dietary linoleic acid has been reported to increase MDA levels and suppress T-AOC in crabs [36], we speculate that the decreased MDA content observed in this study may be associated with reduced linoleic acid levels resulting from enhanced β-oxidation.

E. sinensis muscle represents a high-quality protein source, rich in diverse amino acids that are crucial for nutrition and flavor perception [1,20]. As fundamental units for protein synthesis and key metabolites, amino acids significantly influence the nutritional value and taste profile of aquatic products [37,38]. Dietary lipids can modulate muscle amino acid metabolism [39,40]. In the present study, we observed an increase in glutamic acid (Glu) and lysine, alongside a decrease in proline. Given that Glu contributes umami, lysine contributes bitterness, and proline imparts sweet-umami notes [41,42], this altered profile suggests a potential shift in flavor towards enhanced umami and bitterness with reduced sweetness. To explore the underlying metabolic basis, we investigated key amino acid metabolic genes. The upregulation of gs2, together with gdh, may contribute to nitrogen recycling and glutamate/glutamine balance, potentially supporting the increased Glu and lysine levels and associated flavor changes.

Furthermore, we examined the mTOR signaling pathway, a central regulator of protein synthesis that is known to be modulated by amino acids like Glu and lysine in other aquatic species [43,44]. This pathway promotes translation initiation via phosphorylation of downstream effectors like S6K and 4EBP1 [45]. Unlike reports in teleost fish, we did not observe transcriptional upregulation of mTOR pathway genes or an increase in total protein content in E. sinensis under the same dietary regime. This discrepancy may reflect a potential species-specific difference in the nutrient-sensing mechanisms of crustaceans compared to fish. Overall, the dietary treatment altered the amino acid profile and related flavor compounds, but we found no evidence of a transcriptional response in core mTOR pathway genes or enhanced protein synthesis in E. sinensis muscle under the present conditions.

Lipidomics provides a comprehensive approach to gain deeper insights into the mechanisms of lipid deposition mediated by HOA [46]. Among the differentially expressed lipids, glycerophospholipids (GPs) accounted for the highest proportion at 75.33%, including phospholipids such as PC, phosphatidylethanolamine (PE), and phosphatidylinositol (PI). VIP and ROC analyses identified TG (18:3/18:3/18:3), DG (16:1e/8:0), PC (16:1/19:0), LPC (20:3), and PE (18:0p/20:3) as critical differential lipids following oleic acid supplementation (AUC = 1). Diacylglycerol (DG) serves as a key metabolic intermediate, functioning both as a biosynthetic precursor for TG and glycerol phospholipids (including PC, LPC, and PE), and as a product of TG hydrolysis. The hydrolysis of TG generates DG and free fatty acids (FFA), with the latter being utilized for mitochondrial β-oxidation and ATP production [47]. This metabolic flux aligns with the observed upregulation of genes related to fatty acid β-oxidation in the present study. Pathway enrichment analysis suggested that enhanced glycerophospholipid metabolism is responsible for the elevated levels of PC, LPC, and PE induced by HOA.

As key glycerophospholipids, PC, LPC, and PE play interconnected roles in cell membrane composition, lipid metabolism, signal transduction, and physiological regulation. They undergo enzymatic interconversion, collectively maintaining phospholipid homeostasis. PC, one of the most prevalent phospholipids in cell membranes, is essential for maintaining membrane structure and function, as well as for cell signaling and energy metabolism. Dietary PC supplementation has been reported to improve growth performance and feed utilization in E. sinensis juveniles [48], which is broadly consistent with our findings and supports a potential role of PC in growth regulation. Additionally, PC supplementation has been shown to significantly enhance T-AOC and T-SOD activity while reducing MDA levels [49]. This is consistent with our observation of reduced MDA levels in muscle tissue [50]. LPC, a metabolic derivative of PC, is involved in inflammation and vascular health, with elevated levels potentially linked to pathological conditions [51]. PE is involved in mitochondrial function and cellular processes such as protein folding and autophagy, with potential implications for energy metabolism. Beyond their metabolic and structural functions, lipids also serve as important precursors of flavor compounds [52]. During the cooking process of crabs, phospholipids are prone to oxidation, and the oxidative degradation of lipids and its interaction with the Maillard reaction represent a major pathway for the formation of volatile compounds [53]. Studies have indicated that PE may be a key lipid involved in the generation of aroma substances [54]. However, the specific role of glycerophospholipids in determining flavor profiles still requires further investigation.

The metabolomics and pathway enrichment analyses delineated the remodeling of muscle metabolic profiles in E. sinensis following HOA. The results indicated significant associations between HOA and nucleotide/purine metabolism, linoleic acid metabolism, and the biosynthesis of unsaturated fatty acids. Purine metabolism, a central component of nucleotide turnover, encompasses de novo synthesis, degradation, and salvage pathways. Among its downstream products, GMP and AMP are key determinants of umami taste in crustacean muscle tissue [55]. In this study, HOA did not change muscle GMP or AMP levels, suggesting limited effects on purine-related homeostasis associated with umami formation. This stability may be attributable to the substantial ATP supply generated through fatty-acid β-oxidation, which meets the energetic demands of purine metabolism [56].

Within the inosine monophosphate (IMP) to AMP branch, adenylosuccinate functions as a critical free intermediate in the conversion pathway from IMP to AMP [57]. The decrease in adenylosuccinate may indicate altered flux in this branch; however, unchanged AMP levels suggest that compensatory regulation may help maintain end product homeostasis. Guanosine, a downstream metabolite of GMP involved in energy metabolism and neuroprotection and reported to facilitate glutamate uptake [58], increased in response to OA supplementation. This increase corresponded with the changes observed in glutamate levels.

Regarding lipid metabolism, both DHA and linoleic acid generally depend on exogenous dietary sources [36]. Although enrichment analysis highlighted “unsaturated fatty acid biosynthesis,” linoleic acid decreased while DHA increased. This pattern suggests the presence of compensatory regulation or precursor reallocation triggered by reduced linoleic acid availability. Elevated DHA may contribute to membrane remodeling and activation of regeneration-associated signaling pathways that promote muscle growth [59], consistent with the accelerated growth observed in this study. DHA accumulation has also been associated with increased volatile aromatic compounds [60], suggesting a possible link to flavor improvement.

Overall, HOA may influence growth and flavor quality through integrated changes, including stable purine end-products, shifts in IMP-to-AMP intermediates, and remodeling of polyunsaturated fatty acid profiles. These changes provide potential biochemical explanations for the observed growth improvement and possible flavor enhancement in E. sinensis. However, the proposed mechanisms require further validation through analyses of key enzyme expression and sensory evaluation.

4. Materials and Methods

4.1. Ethics Statement

This experimental protocol was approved by the Animal Care and Use Committee of Nanjing Agricultural University (Nanjing, China). All procedures involving animals were conducted in accordance with the Guideline for the Care and Use of Laboratory Animals in China.

4.2. Experimental Diet Design

Two isonitrogenous and isoenergetic experimental diets were formulated, and their compositions are shown in Table 3. Both diets were based on a commercial fattening-phase diet. In the control diet, soybean oil was used as the added lipid source, whereas in the treatment diet soybean oil was replaced with peanut oil rich in oleic acid (approximately 75%).

4.3. Experimental Animals and Rearing Conditions

The feeding trial was conducted at the Fengbian Village River Crab Farming Cooperative, Gaochun District, Nanjing, Jiangsu Province, China. A total of 160 healthy post-reproductive E. sinensis (initial body weight 135.0 ± 0.45 g) were randomly allocated to two dietary treatments, each with four replicate cages and 20 crabs per cage. Each replicate was reared in a cage (2.0 m × 1.0 m × 0.8 m), and Hydrilla verticillata was planted to cover approximately 20% of the bottom area to provide shelter. After a 1-week acclimation period, crabs were fed the experimental diets for 10 weeks. Crabs were hand-fed twice daily (at 08:00 and 18:00) to apparent satiation; the daily ration corresponded approximately to 1.5–2.0% of body weight. Uneaten feed and feces were siphoned out the following morning, and molting and mortality were recorded daily. Approximately one-third of the water volume in each cage was renewed daily. Throughout the experimental period, water temperature was maintained at 22–28 °C, dissolved oxygen was kept above 5.0 mg/L, pH was maintained between 7.3 and 8.4, and total ammonia nitrogen was kept below 0.05 mg/L by continuous aeration and water exchange.

4.4. Sample Collection

At the end of the feeding trial, crabs with similar dates of reproductive molting (difference ≤ 10 days) were selected and fasted for 24 h, after which final body weight was recorded to calculate weight gain (WG). Hemolymph (approximately 1 mL per crab) was withdrawn from the base of the third walking leg of eight crabs per treatment, centrifuged at 3500× g for 10 min at 4 °C, and the supernatant was collected. Hepatopancreas, gonad, and muscle tissues were excised from eight crabs per treatment to determine the hepatosomatic index (HSI) and gonadosomatic index (GSI). In addition, hepatopancreas, accessory gland, and muscle samples were collected on ice, snap-frozen in liquid nitrogen, and stored at −80 °C for subsequent omics analyses. The indices were calculated as follows:

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4.5. Fatty Acid and Amino Acid Analysis

To profile fatty acids in muscle, samples were hydrolyzed with butylated hydroxytoluene (BHT) as an antioxidant for 1.5 h. After hydrolysis, the reaction was quenched by adding double-distilled water (ddH_2_O), and lipids were extracted with n-hexane. The extracts were centrifuged at 1000 rpm for 10 min, and the upper organic layer was collected for subsequent analysis. Fatty acid composition was then determined using gas chromatography–mass spectrometry (GC–MS; Agilent 7890B–5977A, Agilent, Santa Clara, CA, USA).

Amino acid contents were analyzed following the procedure described previously [61], with minor wording modifications for clarity. Briefly, muscle samples were hydrolyzed in 6 mol/L HCl under a nitrogen atmosphere for 24 h. After hydrolysis, amino acids were quantified using a liquid chromatography analyzer (Agilent 1100, Agilent).

4.6. Biochemical Indicators Analysis

Muscle samples from E. sinensis were collected from each group (n = 8) to determine the activities or levels of alkaline phosphatase (AKP), acid phosphatase (ACP), malondialdehyde (MDA), total antioxidant capacity (T-AOC), catalase (CAT), total superoxide dismutase (T-SOD), glutathione (GSH), triglycerides (TG), total cholesterol (TC), and total protein (TP). All parameters were measured using commercial assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer’s instructions. Specifically, AKP and GSH were determined by the microplate method (Cat. No. A059–2-2 and A006–2-1, respectively); ACP by a spectrophotometric method (Cat. No. A060–1-1); MDA by the thiobarbituric acid (TBA) method (Cat. No. A003–1-1); T-AOC by a colorimetric method (Cat. No. A015–1-2); CAT by the ammonium molybdate method (Cat. No. A007–1-1); T-SOD by the hydroxylamine method (Cat. No. A001–1-1); TG and TC by the microplate method (Cat. No. A110–1-1 and A111–1-1, respectively); and TP by the Coomassie brilliant blue method (Cat. No. A045–2-2).

4.7. Untargeted LC–MS-Based Metabolomic Analysis

For untargeted metabolomics, nine muscle samples were collected from each group, and every three individual samples were combined to generate one pooled biological replicate (three pooled samples per group). Metabolites were extracted by adding 1000 μL of pre-cooled methanol:acetonitrile:water (2:2:1, v/v/v) to each pooled sample, followed by vortex mixing and ultrasonic treatment in an ice bath. After centrifugation, the supernatant was transferred to autosampler vials for LC–MS analysis. A pooled quality control (QC) sample was prepared by mixing equal aliquots of all extracts and was injected periodically throughout the analytical batch to monitor instrument stability.

Chromatographic separation was performed on an Agilent 1290 Infinity UHPLC system (Agilent Technologies, Santa Clara, CA, USA) coupled to a mass spectrometer. Metabolic features with a relative standard deviation (RSD) > 30% in QC samples were removed prior to data normalization and further analysis. Differentially expressed metabolites (DEMs) between groups were screened using partial least squares discriminant analysis (PLS-DA) in combination with univariate statistics, with selection thresholds of variable importance in projection (VIP) > 1, fold change (FC) > 1, and p < 0.05. Metabolite–metabolite associations were evaluated using Pearson correlation coefficients, and overall sample distribution was explored by principal component analysis (PCA). Putatively identified metabolites were annotated and functionally interpreted using the Kyoto Encyclopedia of Genes and Genomes (KEGG) and the Human Metabolome Database (HMDB).

4.8. Untargeted LC–MS/MS-Based Lipidomic Analysis

For lipidomic analysis, nine muscle samples were obtained from each dietary group. To generate composite biological replicates, three individual samples were combined to form one pooled sample, resulting in three pooled replicates per group for LC–MS-based untargeted lipidomics. Lipids were extracted by adding 960 μL of MTBE/methanol (5:1, v/v) to each pooled homogenate, followed by thorough mixing, ice-bath sonication, and centrifugation to achieve phase separation. The resulting upper organic layer was carefully collected, and 75 μL of each extract implied for analysis was dispensed into an LC–MS microplate. Lipid separation and detection were performed using a UHPLC platform (Thermo Fisher Scientific, Waltham, MA, USA) fitted with a Phenomenex Kinetex C18 column and interfaced with a TripleTOF 6600 mass spectrometer (AB Sciex, Framingham, MA, USA). Full-scan MS data were recorded across an m/z window of 200–2000, and MS/MS spectra were acquired in a data-dependent manner from the most abundant precursor ions. The raw files were processed in LipidSearch v4.1 (Thermo Fisher Scientific, Waltham, MA, USA) to conduct peak picking and alignment and to assign putative lipid identities, which were subsequently curated by matching fragment spectra to the LipidBlast library. PLS-DA was applied to evaluate clustering patterns between groups, screen for potential outliers, and confirm dataset consistency. Differential lipid molecules (DLMs) were determined by integrating OPLS-DA outputs with univariate testing. Lipids meeting the thresholds of VIP > 1, FC > 1, and p < 0.05 were considered significantly different between groups.

4.9. RNA Extraction and qRT–PCR Analysis

Total RNA was extracted from eight muscle samples per group using RNAiso Plus (Takara, Dalian, China) in accordance with the supplier’s protocol. To remove potential genomic DNA contamination, the RNA preparations were additionally digested with RNase-free DNase I. RNA quantity and purity were evaluated by spectrophotometry, and only RNA with OD260/OD280 = 1.8–2.1 was used for subsequent experiments. For cDNA synthesis, 1 μg of total RNA from each sample was reverse-transcribed to first-strand cDNA using the PrimeScript™ RT reagent Kit (Takara). qRT–PCR assays were conducted with TB Green™ Premix Ex Taq™ II (Takara). Amplification was performed under the following conditions: 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s and 59–62 °C for 60 s. β-actin served as the reference gene, and relative expression levels were determined using the 2^−ΔΔCt^ approach. Primers were designed from RNA-seq sequences with Primer Premier 5.0 and synthesized by Shanghai Generay Biotechnology Co., Ltd. (Shanghai, China); primer information is provided in Table S1.

4.10. Statistical Analysis

All statistical analyses were conducted in SPSS v23.0 (IBM Corp., Armonk, NY, USA). Prior to hypothesis testing, datasets were examined for normal distribution and variance homogeneity. Differences between the two dietary groups were evaluated using a two-tailed Student’s t-test. Results are reported as mean ± SEM, and p < 0.05 was considered statistically significant.

5. Conclusions

In conclusion, our findings demonstrate that supplementing finishing diets of E. sinensis with oleic acid–rich lipids is an effective and feasible nutritional strategy. The HOA not only promotes growth and improves feed utilization, thereby enhancing overall farming efficiency, but also strengthens antioxidant capacity and modulates lipid and amino acid metabolism in muscle tissue. These changes are expected to contribute to improved nutritional value and flavor quality of crab meat. By integrating multi-omics approaches, this study elucidated the metabolic networks and potential regulatory pathways through which HOA acts on muscle quality in E. sinensis. These results provide a theoretical basis for the precise application of HOA in aquaculture practices aimed at optimizing product quality and offer valuable insights for the rational use of functional lipids in crustacean nutrition and breeding.