Evaluation of Nutritional Value of the First Generation (G1) of Population Breeding of Eriocheir sinensis “King Crab 1”

Abstract

The nutritional composition of commercially valuable crabs is governed by a complex interplay of hereditary factors, growth environment, developmental stage and feed composition. In this study, the nutritional characteristics of edible tissues were quantitatively compared among four populations of Eriocheir sinensis: a non-selected group (NF), the G1 generation of the selective bred “King Crab 1” group (SF), the SF group with water quality regulated using microbiological agents (SF8) and a chilled-fish feeding group (CF). Growth metric analysis revealed that females in the SF group exhibited slightly superior growth performance compared to other groups. Amino acid analysis demonstrated that compared to the NF group, the content of essential and umami amino acids in the ovary was remarkably increased in the SF8 group. Additionally, the SF and CF groups exhibited elevated contents of flavor amino acids in male crabs. Moreover, the CF group exhibited the highest contents of EPA and DHA, and the highest n-3/n-6 PUFAs ratio, with the SF group following. Overall, although the G1 generation of selectively bred crabs demonstrated improved nutritional indicators compared to the unselected group, they still lagged behind the CF group in several aspects. These findings provide valuable insights and data support for future breeding strategies.

1. Introduction

The Chinese mitten crab (Eriocheir sinensis), a decapod crustacean within the family Varunidae, demonstrates obvious feeding and reproductive migratory behaviors, with major populations primarily inhabiting the Yangtze, Liaohe, and Oujiang River basins [1,2]. With unique flavor and appealing aroma, E. sinensis possesses high nutritional value, characterized by an abundance of essential amino acids (EAA), unsaturated fatty acids, and bioavailable micronutrients, which are appreciated by consumers [3,4]. Official statistics from China’s Bureau of Fisheries and Fishery Administration estimated that the yield of cultured E. sinensis reached 888,629 tons in 2023, of which 405,460 tons, approximately 45.63% of the national total, were produced in Jiangsu Province [5]. However, the prevalent use of unscientific feeding practices, low quality feed, and extensive reliance on chilled fish in aquaculture has led to environmental degradation and inconsistent production quality. These issues have significantly impeded industry sustainability, underscoring the necessity for a comprehensive nutritional evaluation of this economically significant species.

The nutritional profile of economic crab tissues serves as a key determinant of both nutritional value and market appraisal [6]. The dynamics of nutrient deposition in edible tissues, particularly with regard to lipids and proteins, are primarily influenced by the status of gonadal maturation [7]. Current nutritional assessments of E. sinensis have primarily focused on proximate composition (protein, lipid, moisture, ash), essential amino acids, fatty acid profiles, and mineral content. The nutritional value of E. sinensis is driven by various factors, e.g., genetic background, habitat environment, developmental stage and feeding regimen [7]. Comparative analyses between the newly bred varieties “Jianghai 21” and “Changjiang 2” revealed that crude lipids were more abundant in “Changjiang 2”, whereas “Jianghai 21” demonstrated elevated levels of total amino acids (TAA) and EAA, indicating significant variations in nutrient composition among newly developed strains [8]. Aquaculture practices have been shown to significantly influence the nutritional profile of E. sinensis. Compared to paddy-reared E. sinensis, the wild-caught E. sinensis exhibited superior flavor profiles and higher concentrations of umami amino acids (UAA), minerals, monounsaturated fatty acids (MUFAs) and polyunsaturated fatty acids (PUFAs) [9]. Co-cultivation of Yangtze and Liao River crabs in Jilin’s paddy fields (a major production area of Liao crabs) revealed distinct nutritional characteristics: Liao crabs exhibited higher crude protein and lipid content with optimal amino acid composition in edible tissues, while Yangtze crabs displayed greater PUFAs accumulation in the gonads. These findings underscore the need for targeted strategies to preserve the nutritional integrity of “Southern crabs cultivated in the North” [10]. Additionally, a study by Wang et al. on E. sinensis from Ya Lake in Qinghai, China, demonstrated that female crabs exhibited peak levels of C20: 5n-3 (Eicosapentaenoic acid, EPA) in the hepatopancreas, as well as glutamic acid (Glu) and arginine (Arg) in the ovary in August. By September, both sexes exhibited maximal total lipid content in the hepatopancreas and gonad, along with elevated levels of alanine (Ala) and Glu in muscle tissue, demonstrating clear temporal dynamics in tissue-specific nutrient composition during the developmental cycle of E. sinensis [11]. Dietary lipid levels were found to significantly modulate the fatty acid profile in the hepatopancreas. Specifically, total unsaturated fatty acid content increased significantly at a lipid level of 13%. In contrast, a high lipid level of 16% inhibited fatty acids accumulation [12]. In summary, the multifactorial influences of different varieties, culture systems, and feeding strategies on the nutrient composition of E. sinensis provide a robust theoretical basis for optimizing breeding protocols and enhancing aquaculture productivity.

To meet the market demand, aquatic breeders have devoted substantial efforts to the artificial selection and breeding of E. sinensis, successfully developing several nationally recognized new varieties. However, in recent years, the germplasm resources of E. sinensis in the Yangtze River have experienced gradually degraded, with a particularly noticeable decline in size and flavor [13]. Current breeding programs are primarily targeted at enhancing growth rates and increasing the proportion of large-sized individuals, resulting in a lack of diversity in breeding objectives, which makes it challenging to satisfy the growing market demand for high-quality crabs. Therefore, advancing the synergistic genetic improvement of multiple traits in E. sinensis to achieve comprehensive varietal optimization represents a critical research priority. To address these challenges, our research team initiated a selective breeding program in 2022. This program utilized foundation stocks from cultured populations in Suqian and Yixing (Jiangsu Province), and a wild population from Luoma Lake in Suqian, to develop a new E. sinensis strain, “King Crab 1”, which has now advanced to the G2 generation. The core breeding objectives for this strain encompass not only the enhancement of growth performance and tolerance to high-temperature stress, but also the assurance of its nutritional quality. When reared exclusively with formulated feed throughout the entire growth period, the nutritional quality of these crabs is comparable to that of crabs fed the traditional chilled fish. Consequently, this study was designed to conduct a systematic evaluation of the nutritional value of the G1 generation of this strain under different culture environments and feeding regimes, thereby providing a scientific basis and guidance for subsequent breeding efforts.

2. Materials and Methods

2.1. Sample Collection

The E. sinensis button crabs used in the experiment were obtained from the Canal Bay aquaculture farm of the Suqian Institute of Agricultural Science, Jiangsu Academy of Agricultural Sciences (33.97° N, 118.32° E). The study was conducted from March 2023 to November 2024, and included four experimental groups: (1) non-selected population + feed (NF group), (2) selected G1 generation (King Crab 1) + feed (SF group), (3) selected G1 generation (King Crab 1) + feed + microbial agent T8 (Shandong Maia Biotechnology Co., Qingdao, China, SF8 group), and (4) non-selected population + chilled fish feeding (CF group). The experimental design permits three key comparisons to delineate the specific effects of selective breeding, microbial supplementation, and their combined impact. First, the effect of selective breeding is assessed by contrasting the NF and SF groups, thereby isolating the genetic contribution to trait enhancement under standardized dietary conditions. Second, the efficacy of the microbial agent is evaluated by comparing the SF group with the SF8 group, a contrast which reveals any additive effect of the supplement within the improved genetic background. Finally, the combined benefit of the modern breeding and feeding strategy over the traditional practice is determined by comparing the CF group with both the SF and SF8 groups, which serves to quantify the overall advantage of the integrated modern system. Unless otherwise specified, all analyses described in this study were performed with three biological replicates per sample type per group (n = 3).

The microbial agent (T8) utilized in this trial contained Bacillus subtilis and Pseudomonas fluorescens as its primary constituents, with a viable bacterial count of ≥10⁹ CFU·mL⁻¹. The product is designed with a dual of action: to ameliorate water and sediment quality by converting harmful substances such as ammonia nitrogen and nitrite, and to enhance crab immunity and growth performance by suppressing pathogenic bacteria (e.g., Vibrio spp.) and modulating the host’s intestinal microbiota. The application protocol was defined as a dosage of 1.8 L per 400 m², administered monthly, with frequency increased to twice per month during the high-temperature period (July to August).

All experimental ponds were outdoor earthen ponds with an area of approximately 400 m². Stocking density (2.1 crab/m²) and management practices were standardized across all groups, with no significant differences detected in initial morphometrics of the button crabs (NF&CF: 9.83 ± 0.97 g, SF&SF8: 9.87 ± 1.05 g). All experimental crabs were fed daily with a commercial crustacean feed (Guangdong Haida Feed Co., Ltd., Guangzhou, China) containing 25% fish meal, 18% soybean meal, 25% wheat flour, 10% cottonseed meal, 8% corn gluten meal, 5% rapeseed meal, 3% krill oil, 2% squid paste, 3% calcium dihydrogen phosphate, 0.5% vitamin premix, and 0.5% mineral premix, administered at 3% of their live biomass. The approximately nutrient composition of the feed and chilled fish was shown in

Table 1. The approximate nutrient composition of fed and chilled fish (n = 3).

Feeding	Moisture (% Wet Weight)	Crude Protein (% Dry Weight)	Crude Lipid (% Dry Weight)	Ash (% Dry Weight)
Feed	10.39 ± 0.51	44.67 ± 0.70	9.39 ± 0.21	11.98 ± 0.47
Chilled-fish	79.18 ± 0.40	56.85 ± 0.72	20.39 ± 0.93	21.94 ± 0.63

.

2.2. Growth Performance

At the end of November 2024, nine crabs of both sexes were randomly collected from each experimental pond. After surface moisture was removed, the crabs underwent biometric analysis (0.01 g precision for weight; 0.01 mm precision for carapace width and length measurements). Following dissection, hepatopancreatic, gonadal, and muscular tissues were weighed (0.01 g precision) for gravimetric quantification. The hepatopancreatic index (HSI, %), gonadal index (GSI, %), meat yield (MY, %), total edible yield (TEY, %) and conditional factor (CF, g/cm³) were calculated using the following formulas [14]:

HSI (%) = hepatopancreatic weight/body weight × 100%,

(1)

GSI (%) = gonadal weight/body weight × 100%,

(2)

MY (%) = muscular weight/body weight × 100%,

(3)

TEY (%) = HSI + GSI + MY,

(4)

CF (g/cm³) = body weight/carapace length³.

(5)

2.3. Determination of Proximate Composition

The proximate composition of hepatopancreas, gonad, and muscle of across experimental groups was analyzed following Chinese national standard methodologies. Moisture content was quantified through atmospheric pressure drying at 105 °C in an oven (GB/T 10358-2008) [15]. For the determination of crude ash content, the samples were first carbonized to smokeless by an electric pottery furnace, burnt in a muffle furnace at 550 °C for 4 h to a constant weight, and then weighed to calculate the crude ash content (GB/T 6438-2007) [16]. Assessment of crude protein content was carried out according to Kjeldahl method (GB/T 6432-2018) [17]. A constant weight dry sample with mixed catalyst and sulfuric acid was first placed in a Kjeldahl flask and decocted on an electric stove. When the decoction is cooled, alkaline distillation to free ammonia is absorbed with boric acid absorbent solution and then titrated with hydrochloric acid standard solution, and the protein content is calculated according to the consumption of acid. The crude lipid in the samples was extracted by petroleum ether using a fully automated Soxhlet ether extractor, and the ether was distilled off and weighed to obtain the crude lipid content (GB/T 6433-2006) [18].

2.4. Evaluation of Amino Acid Composition and Content

The amino acid content of the samples was assayed using the National Standard for Food Safety (GB 5009.124-2016) [19]. Specifically, lyophilized samples of hepatopancreas, gonad and muscle tissues (20 mg) were subjected to acid hydrolysis using 10 mL of 6 mol/L HCl in sealed reaction vessels. The hydrolysis tube was frozen in a freezing agent for 5 min, then evacuated and filled with nitrogen, and repeated three times. After hydrolysis at 110 °C for 22 h in a hydrolysis furnace, the powder was dried under reduced pressure using a parallel evaporator. Then, 1.0 mL of sodium citrate buffer (pH 2.2) was added, and the mixture was filtered through a 0.22 μm aqueous filter membrane. Subsequently, the amino acid content was determined and calculated using an ultra-high-speed amino acid analyzer (HITACHI LA8080, Tokyo, Japan) with reference to the standard.

2.5. Mineral Element Analysis

Elemental analysis was performed using inductively coupled plasma optical emission spectrometry (ICP-OES; Jena PQ9000, Jena, Germany) for quantification of K, Ca, Na, Mg, P, Fe, Mn, Cu, and Zn concentrations in hepatopancreas, gonad, and muscle tissues, while Se quantification was conducted via ICP-mass spectrometry (Thermo iCAPTM Q, Waltham, MA, USA). Referring to the method of Pinto et al. [20], 0.4 g of dry powder sample was accurately weighed, and 7.5 mL of 65% nitric acid (HNO₃) and 2.5 mL of 37% hydrochloric acid (HCl) were added to dissolve the samples. The digestion process was divided into three steps: the temperature was increased from room temperature to 90 °C within 30 min, maintained at this temperature for 30 min, and then held at 105 °C for 60 min. After cooling, the sample solution was diluted to 25 mL with deionized water, filtered, and stored in labeled tubes for on-board assay.

2.6. Analysis of Fatty Acids

Tissue samples (hepatopancreas, gonad, and muscle) were collected from three crabs per group. For each crab, the three tissues were pooled to form a single composite sample per individual, resulting in three composite samples per group (n = 3). The fatty acid relative content of these composite samples was then quantified following the National Standard for Food Safety (GB 5009.168-2016) [21]. Briefly, after accurately weighing the dry powder of edible tissues samples, 4 mL of iso-octane was added to dissolve the samples. Subsequently, 200 µL of potassium hydroxide-methanol solution was added and mixed thoroughly, allowing the solution to clarify before adding 1 g of sodium bisulfate and mixing well to neutralize the excess potassium hydroxide. After the salt had precipitated, the upper solution was transferred to a sample vial and measured using a gas chromatograph (Agilent 7890B, Santa Clara, CA, USA) with a hydrogen flame ionization detector (FID).

2.7. Statistical Analysis

Data are expressed as mean ± standard deviation (S.D.). The normality of distribution and homogeneity of variance were assessed using the Shapiro–Wilk test and Levene’s tests, respectively. Intergroup differences were evaluated by one-way ANOVA followed by Tukey’s post hoc test (SPSS 25.0; IBM Corp., Chicago, IL, USA). Statistical significance was set at p < 0.05, with highly significant differences defined as p < 0.01.

3. Results

3.1. Growth Assessment

In females, the SF group showed a markedly higher body weight compared to the NF group (p < 0.01) (

Table 2. Growth parameters of male and female E. sinensis in different experimental groups.

Group	Female				Male
	NF	SF	SF8	CF	NF	SF	SF8	CF
Body weight (g)	100.71 ± 25.71 a	153.38 ± 41.64 b	137.99 ± 24.11 ab	125.15 ± 29.01 ab	135.58 ± 39.91 a	143.04 ± 43.32 a	166.83 ± 18.21 ab	205.47 ± 52.68 b
Carapace length (mm)	57.11 ± 6.03 a	66.89 ± 7.04 b	65.78 ± 4.12 b	62.00 ± 4.92 ab	58.83 ± 4.92 a	63.44 ± 5.81 ab	64.56 ± 2.30 ab	68.00 ± 4.85 b
Carapace width (mm)	60.89 ± 5.86 a	69.11 ± 5.67 b	70.44 ± 4.56 b	66.78 ± 4.63 ab	63.17 ± 4.79 a	67.44 ± 7.63 ab	71.22 ± 3.31 b	74.56 ± 5.03 b
Conditional factor (g/cm3)	0.53 ± 0.04	0.50 ± 0.05	0.48 ± 0.04	0.52 ± 0.05	0.65 ± 0.06 b	0.55 ± 0.07 a	0.62 ± 0.06 ab	0.64 ± 0.06 b

Note: Data are expressed as wet weight (n = 9). Different letters represent significant differences among the different treatments in the same sex (p < 0.05).

), while the differences were not significant when comparing the SF group with the SF8 or CF groups (p > 0.05). Compared to the NF group, both the SF and SF8 groups exhibited significantly greater carapace length and width (p < 0.05). However, comparisons of the condition factor revealed no significant intergroup differences (p > 0.05). In males, compared to the NF and SF groups, the CF group showed significantly increased body weight (p < 0.05). Similarly, the carapace length in the CF group was significantly greater than that in the NF group (p < 0.01). Furthermore, compared with the NF group, significantly larger carapace width was observed in the CF and SF8 groups (p < 0.05). The condition factor varied among the four groups, with the CF and NF groups demonstrating significantly elevated levels compared to the SF group (p < 0.05). For females, the CF group showed significantly greater GSI than other groups (p < 0.05), while no significant differences were observed in HSI, MY, or TEY (p > 0.05) (Figure 1). In male individuals, the highest HSI values were recorded in the SF group, showing significant differences from the NF group (p < 0.01). The MY was significantly higher in the CF group compared to the SF group (p < 0.05). Additionally, GSI and TEY levels did not differ significantly between male treatment groups (p > 0.05).

3.2. Approximate Compositional Analysis of Edible Tissues

The approximate composition of edible tissues was presented in Figure 2. In female crabs, the SF, SF8 and CF groups all exhibited significantly lower hepatopancreatic moisture content compared to the NF group (p < 0.05). Conversely, the SF and SF8 groups showed significantly lower crude protein levels than both NF and CF groups (p < 0.05). Ovarian crude protein content demonstrated marked variation, with the SF group showing significantly lower levels than the NF, SF8, and CF groups. Moreover, the CF group displayed the highest ovarian crude protein content among all experimental groups (p < 0.05). Similarly, muscle crude protein content was highest in the CF group, and significantly different from that of the SF and SF8 groups (p < 0.01). Tissue-specific variations in crude lipid content were observed in female crabs (hepatopancreas > gonad > muscle), with the SF group presenting maximal accumulation in all tissues. Concretely, hepatopancreatic crude lipid content in the SF group was significantly higher than that in the SF8 group (p < 0.05). In contrast, the CF group showed significantly lower crude lipid content in both gonad and muscle compared to the other three groups (p < 0.05).

Among males, moisture content in the gonad was significantly higher in the SF group compared to the NF and CF groups, with the SF8 group showing the highest values overall (p < 0.01). Both ash and crude protein contents in edible tissues varied significantly among the groups (p < 0.05). Specifically, hepatopancreatic ash content peaked in the NF group and was significantly greater than that in the SF8 and CF groups (p < 0.05). Ash content in gonad exhibited treatment-specific variation, with the SF group having higher levels than the SF8 group, while the CF group exhibited significantly higher values than both the NF and SF8 groups (p < 0.05). In muscle, ash content showed significantly higher levels in the NF and SF groups compared to the SF8 and CF groups (p < 0.05), with the NF group being the highest value (p < 0.05). Regarding crude protein content, the CF group had the highest hepatopancreatic levels, significantly exceeding those of the other three groups (p < 0.05). The SF group also exhibited significantly higher levels than the SF8 group (p < 0.05). In gonad, the CF group displayed significantly greater crude protein content than the NF and SF8 groups, and the SF group showed significantly elevated levels compared to the NF group (p < 0.05). Likewise, crude protein content in muscle was significantly higher in both the CF and SF groups than in NF and SF8 groups (p < 0.05). In contrast, the crude lipid content of the gonad was significantly higher in the NF and SF8 groups compared to the SF and CF groups (p < 0.01).

3.3. Amino Acid Composition

The amino acid composition of the hepatopancreas, gonad and muscle tissues in E. sinensis was shown in Figure 3. In the hepatopancreas, a significantly greater essential to non-essential amino acids ratio (E/N) was observed in female SF group relative to NF group (p < 0.05). In the gonad, amino acid content varied considerably across four groups. Specifically, in female individuals, the SF8 group exhibited significantly higher levels of Lys, Ile, Leu, Glu, Cys, EAA and UAA than the NF group (p < 0.05). Additionally, the E/N ratio in the ovary was significantly higher in the SF8 group than in both the NF and CF groups, and it was also higher in the SF group than in the NF group (p < 0.05). Similarly, the CF group showed significantly elevated levels of Asp, Ser, Pro, and UAA compared to the NF group (p < 0.05). Compared to the SF group, the SF8 group had significantly higher contents of Lys, Glu and Cys, while the CF group showed significantly increased levels of Asp, Ser and Pro (p < 0.05). Moreover, Pro content was significantly higher in the CF group than in the SF8 group (p < 0.01). In male gonad, apart from Met, His, Arg, Asp, Gly, Cys, Pro, and the ratio of essential to total amino acids (E/T) and E/N, the remaining amino acids and related indicators were significantly higher in the SF and CF groups compared to the NF and SF8 groups (p < 0.05). The CF group exhibited significantly greater contents of Phe, Thr, Leu, Ala, Glu, Ser, Cys, Tyr, TAA, UAA, EAA, non-essential amino acids (NEAA), sweet amino acids (SAA), and bitter amino acids (BAA) than the SF group (p < 0.05). Furthermore, Cys content in the CF group remained significantly higher even when compared to the SF8 group (p < 0.01). In contrast, the CF group had the lowest E/T and E/N ratio, which were significantly lower than those in the other three groups (p < 0.05), while the SF group had a significantly lower E/N ratio than the NF group (p < 0.05). The SF8 group exhibited comparable amino acid content to the NF group across all classifications, with no statistically significant differences (p > 0.05). In female muscle, the SF and SF8 groups demonstrated significantly higher Ile and Val contents compared to the NF group (p < 0.05), whereas the CF group exhibited markedly elevated Pro content compared to all other experimental groups (p < 0.01). In contrast, no significant differences in amino acid content were observed in male muscle tissues across the four groups (p > 0.05). However, the E/T and E/N ratios were significantly higher in the SF and SF8 groups than in the NF and CF groups in both sexes (p < 0.01).

Figure 2. Analysis of the proximate composition of the edible tissues of E. sinensis in different experimental groups. Data for moisture content is expressed on wet weight basis, while crude protein, crude lipid and ash contents are expressed on dry weight basis (n = 3). Different letters indicate significant differences among different groups in the same tissue (p < 0.05).

Figure 2. Analysis of the proximate composition of the edible tissues of E. sinensis in different experimental groups. Data for moisture content is expressed on wet weight basis, while crude protein, crude lipid and ash contents are expressed on dry weight basis (n = 3). Different letters indicate significant differences among different groups in the same tissue (p < 0.05).

Figure 3. Amino acid content of hepatopancreas (A), gonad (B) and muscle (C) of different experimental groups of E. sinensis. EAA is the essential amino acid, NEAA is the non-essential amino acid, UAA is the umami flavor amino acid, SAA is the sweet flavor amino acid, BAA is the bitter flavor amino acid, TAA is the total amino acid. E/T refers to the ratio of the essential amino acid to the total amino acid, and E/N refers to the ratio of the essential amino acid to the non-essential amino acids to total amino acids, and E/N refers to the ratio of essential to non-essential amino acids. Data are presented in log₁₀(value + 0.01) and expressed on dry weight basis (n = 3). Different letters for the same sex indicate significant differences among different groups (p < 0.05).

Figure 3. Amino acid content of hepatopancreas (A), gonad (B) and muscle (C) of different experimental groups of E. sinensis. EAA is the essential amino acid, NEAA is the non-essential amino acid, UAA is the umami flavor amino acid, SAA is the sweet flavor amino acid, BAA is the bitter flavor amino acid, TAA is the total amino acid. E/T refers to the ratio of the essential amino acid to the total amino acid, and E/N refers to the ratio of the essential amino acid to the non-essential amino acids to total amino acids, and E/N refers to the ratio of essential to non-essential amino acids. Data are presented in log₁₀(value + 0.01) and expressed on dry weight basis (n = 3). Different letters for the same sex indicate significant differences among different groups (p < 0.05).

3.4. Mineral Elements

As illustrated in Figure 4, in female hepatopancreas, K content in the NF group was significantly elevated compared to the SF8 group (p < 0.01). In male hepatopancreas, Na and Mg contents exhibited significant intergroup variation. Specifically, the NF group showed markedly higher Na and Mg levels compared to the SF and CF groups (p < 0.05). Additionally, Na content in the SF8 group was significantly higher than that in the CF group (p < 0.05). In the ovary, significant differences in macro-elements except for K and Na were observed across experimental groups (p < 0.05). The SF group exhibited significantly elevated Ca levels compared to the NF and CF groups. Meanwhile, the NF and SF8 groups had higher Mg and P contents than the SF group (p < 0.05). Furthermore, the content of P in the SF8 group was significantly higher than that in the SF group (p < 0.05). However, opposite trends were presented in the testis. K content was significantly lower in the CF group compared to the SF and SF8 groups, and the content of P was significantly higher in the NF and SF groups than in the CF group (p < 0.05). In female muscle, the SF group showed significantly increased Mg levels compared to the NF group (p < 0.05). Likewise, the content of Ca was highest in the SF group, and significantly higher than that in the NF and CF groups. The SF8 group also had significantly higher Ca levels than the NF group (p < 0.05). Within male muscle, no statistically significant differences were observed in macronutrient concentrations across groups (p > 0.05), with the exception of Mg, which demonstrated significantly reduced levels in the CF group compared to other experimental groups (p < 0.05).

As for trace element contents, Cu content in the CF group of female hepatopancreas was significantly higher than that in the NF and SF8 groups (p < 0.05) (Figure 5). Ovarian Zn concentration was significantly elevated in the NF group compared to the SF group, while Cu levels in the NF and CF groups were markedly elevated compared to those in the SF and SF8 groups (p < 0.05). In the testis, the content of Cu was significantly higher in the CF group than in the NF and SF8 groups, and also higher in the SF group than in the NF group (p < 0.05). Additionally, Fe content in female muscle was significantly higher in the SF group than that in the CF group (p < 0.01). And the Cu content in the CF group was significantly higher than that in the other three groups (p < 0.05). Mn content in the SF group was significantly higher than that in the SF8 and CF groups (p < 0.05), while the Se content in the CF group was significantly higher than that in the NF group (p < 0.05). In male muscle, only Fe content showed significant intergroup variation, with the SF group exhibiting significantly higher Fe content than CF group (p < 0.05).

3.5. Fatty Acid Relative Content Analysis

A total of 25 known fatty acids were detected in the edible tissues of E. sinensis (Figure 6). The CF group exhibited the highest level of saturated fatty acids (SFAs), MUFAs, and polyunsaturated fatty acids (including n-3, n-6, and n-3/n-6 PUFAs), whereas the SF group showed the lowest content of SFAs and MUFAs. The NF group contained the lowest amounts of PUFAs (n-3, n-6, and n-3/n-6 PUFAs), however, none of these differences reached statistical significance when compared to the other groups (p > 0.05). Regarding specific fatty acid relative content, both the SF and SF8 groups had significantly higher levels of C12:0 compared to the NF group, while the CF group showed elevated C14:0 content (p < 0.05). Furthermore, the SF group exhibited significantly increased C18:3n-6 level compared to the NF group, although its C18:0 content was significantly lower than that of the CF group (p < 0.05). Notably, the NF group demonstrated the lowest C14:1n-5 level among all groups, whereas the SF8 group exhibited the highest C20:0 content (p < 0.05). Remarkably, the CF group outperformed the NF group in several key parameters, displaying significantly higher concentrations of C20:5n-3 (EPA), C22:6n-3 (DHA) and their combined total (p < 0.01). Moreover, the CF group showed a higher DHA/EPA ratio than both the SF and SF8 groups and maintained significantly elevated n-3/n-6 PUFAs ratios compared to the NF and SF8 groups (p < 0.05).

4. Discussion

This study illustrates that selective breeding has a notable sex-specific impact on the growth of the E. sinensis. Specifically, selective breeding (SF&SF8 groups) significantly improved female morphometric parameters (body weight, carapace length, and carapace width) compared to the NF group, while no discernible selection effects were observed in males. This finding indicates that our selective breeding strategy has already yielded significant growth advantages for females in the current G1 generation. Thus, subsequent breading efforts should focus on targeted improvements of male growth traits. In terms of diet, compared with the CF group, the body weight of males in the NF and SF groups was significantly lower, while no significant difference was found in the SF8 group, indicating that the microbial agent may partially mitigate the growth-limiting effects of compound feed on male individuals, although this effect has not yet reached statistical significance. Differences in growth traits essentially represent the external manifestation of an organism’s energy allocation strategy [22]. Analysis of the proportion of primary edible tissue provides intrinsic evidence for this. Our findings revealed significantly higher GSI values in females from the CF group compared to other experimental groups, along with slightly reduced HSI. This pattern aligns with the typical characteristic of the rapid gonadal development phase in females, where energy and nutrients are preferentially directed towards gonadal enrichment [23]. In contrast, male individuals in the selection groups (SF group) exhibited significantly elevated HSI and markedly lower MY compared to the NF group, which consistent with the aforementioned reduction in male body weight. This indicates that during the same rearing stage, the energy allocation in males remains primarily directed toward the development of the hepatopancreas, with gonadal development lagging significantly behind that of females. This temporal discrepancy is likely driven by the combined effects of the reproductive cycle and endocrine regulation [24]. In crustaceans, ecdysones (such as 20-hydroxyecdysone) and gonad-suppressing hormones synergistically regulate resource allocation between growth and reproduction [25,26,27]. Female crabs may enter reproductive maturity earlier, with metabolic resources prioritized towards gonadal development to support yolk synthesis; whereas male crabs defer material and energetic investment in reproduction, channeling more energy towards body growth and preparation for subsequent reproductive competition [28]. This physiological mechanism explains why selective breeding yields more direct and pronounced effects on female crab growth and gonadal development.

Differences in energy allocation among organs further drive significant differentiation in the composition of edible tissue. Analysis of the approximate composition of edible tissues revealed that selective breeding and dietary intervention influenced the metabolic pathways and edible quality of E. sinensis by regulating the deposition patterns of fundamental nutrients. Moisture is an essential quality indicator that affects the stability of edible tissues in aquatic products. It regulates microbial activity and determines the types of microorganisms present, thereby effectively reflecting shelf-life and critically influencing the development of microorganisms and spores [29,30]. In female hepatopancreas, moisture content was significantly reduced in the SF, SF8, and CF groups compared to the NF group, indicating that selective breeding and the feeding of chilled fish potentially extending the shelf-life of the hepatopancreas in female crabs. In contrast, male gonads demonstrated significantly higher moisture content in the selected breeding groups, reflecting a potential reduction in the shelf-life of the “crab butter”, which was further exacerbated by the use of microbiological agent. Ash content primarily consists of mineral salts, and its level directly reflects the deposition of essential minerals such as Ca, Mg and P, which are crucial for the development of the exoskeleton of crabs [31]. Ash analysis indicates that selective breeding promotes mineral deposition in male crab muscle tissue, whilst chilled fish more effectively enhances mineral accumulation within the gonads. More crucially, the composition of protein and lipid exhibits distinct gender-specific distribution patterns. The female crab selection groups (SF and SF8) exhibited a “high-fat, low-protein” profile which was consistent with the increase in the GSI index in female crabs, indicating that females in the SF group may have been in a gonadal development stage where lipids served as the primary energy source. This suggests that their metabolic pattern favored prioritizing energy allocation to the gonadal tissues [7,23]. Male crabs, on the other hand, showed a distinct nutrient allocation strategy. The crude protein content in the CF group was highest across all three edible tissues, coupled with lower crude fat levels in the gonads This overall nutritional profile of ‘low fat and high protein’ indicates superior edible quality. The sexual dimorphism in nutrient allocation fundamentally reflects divergent reproductive strategies. Female crabs require substantial lipids as raw material and energy for yolk production, hence exhibiting a “high-fat” metabolic bias during rapid gonadal development [32]. Conversely, male reproductive success relies more on muscular function and mating behavior, leading to a “high-protein” nutritional profile [24]. The roles of selective breeding and diet essentially amplify or modulate these hormone-mediated sex-specific metabolic programs.

The metabolic flow driven by energy allocation further determines protein synthesis efficiency and amino acid composition patterns. Overall, the amino acid composition of E. sinensis remained stable in the hepatopancreas, with no significant differences observed between groups. This is consistent with the findings of Feng et al. [33], i.e., that no significant differences in amino acid composition were observed in edible tissues of E. sinensis fed either formulated diets or chilled fish. This suggested that amino acid composition gradually tends to be stable and is less influenced by diet or environment in the course of long-term aquaculture and domestication. However, the amino acid composition in the gonad varied significantly across the experimental groups, with greater variation observed in male gonad than in female gonad. This is agreement with the fact that male crabs develop later than females, and by the time female gonad is fully developed, their nutrient composition tends to stabilize [28]. This study further revealed that the breeding strategy significantly enhanced the E/N ratios across the entire female E. sinensis (including hepatopancreas, gonad and muscle), which is in accordance with the aforementioned significant increase in crude fat content in female crabs, indicating that female crabs in the selected breeding groups exhibited a more balanced amino acid composition and a substantial improvement in protein quality [34]. In contrast, microbial agent (SF8 group) exerted no significant influence on amino acid content. Among male individuals, the E/N ratio in the gonads of the selected breeding group actually decreased, primarily due to increased levels of non-essential amino acids. This reveals active amino acid metabolism and restructuring processes occurring during the delayed gonadal development phase in male crabs [35]. On the other hand, the amino acid composition in the selection breeding groups (SF and SF8 groups) was superior to that in the CF group. This finding holds significantly practical value, as by optimizing the selection strategy, the high-cost proportion of chilled fish in feed can potentially be reduced in future breeding programs while maintaining or even improving the nutritional quality and growth performance of E. sinensis. Apart from nutritional quality, flavor characteristics equally influence commercial value. Regarding flavor amino acids, feeding chilled fish demonstrated a traditional advantage, with both this group and the microbial preparation group significantly increasing umami amino acid content in female crab gonads. The contents of UAA, SAA and BAA in the male gonads were significantly elevated in the SF and CF groups compared to both the NF and SF8 groups, with UAA and SAA predominating. Although no statistically significant differences were observed between the selected breeding group and chilled fish group in core amino acids contents, the former group was still slightly inferior to the chilled fish group in terms of the enrichment of the flavor-enhancing amino acids. This represents a trait requiring attention and improvement in future high-quality breeding programs. Overall, the breeding efforts successfully conferred a more balanced essential amino acid profile upon female crabs; however, chilled fish remain a valuable reference for optimizing flavor compounds.

The fatty acid profiles—particularly the relative content of PUFAs—constitute another core dimension defining the nutritional and health value of E. sinensis [14]. As an important source of high-quality essential fatty acids, the PUFAs content of E. sinensis serves as a core indicator for evaluating its nutritional value [36]. This study reveals that selective breeding and dietary exerted promising effects on the relative content of PUFAs in E. sinensis. All groups of E. sinensis exhibited high PUFAs ratios (NF: 31.05%, SF: 48.18%, SF8: 47.74%, and CF: 49.08%), with the relative PUFAs proportion approaching 50% in the selected breeding groups (SF group) and the CF group. This high PUFAs profile renders E. sinensis a potential functional food for the prevention of inflammatory and degenerative diseases. Epidemiological and intervention studies have confirmed the preventive effects of PUFAs against various diseases, including cardiovascular disease, rheumatoid arthritis and asthma [32,37]. Crucially, although the relative content of n-3 and n-6 PUFAs showed no significant differences between groups, the microbial preparation (SF8 group) markedly enhanced the n-3/n-6 PUFA ratio. This optimization is pivotal for exerting anti-inflammatory effects and promoting cardiovascular health. The n-3 and n-6 PUFAs and their ratio (n-3/n-6 PUFAs) are also recognized as critical factors influencing human health. And this ratio not only plays an essential function in the pathogenesis of cardiovascular diseases, but is also closely relate to cancer, inflammation and autoimmune diseases [38]. A lower n-3/n-6 PUFAs ratio may have adverse health effects, whereas a higher ratio can significantly increase the nutritional and commercial value of aquatic products [34]. Nevertheless, analysis of the two key long-chain PUFAs—EPA and DHA—revealed differences in efficacy between the strategies. The incorporation of EPA and DHA into cellular tissues can influence membrane fluidity and permeability, as well as the activity of transmembrane proteins [39]. Both EPA and DHA have been shown to regulate K⁺, Na⁺ and Ca²⁺ channel activity in cardiac myocytes, thereby modulating the electrical excitability and contractility of myocytes in relation to growth, reproduction and neurodevelopment [40,41,42]. However, analysis of the two key long-chain PUFAs—EPA and DHA—revealed differences in efficacy between the strategies. In the present study, the CF group demonstrated a significant advantage in effectively enhancing the DHA and EPA relative contents. Whilst the selected breeding groups (SF and SF8 group) exhibited improvement compared to the NF group, this improvement was not statistically differences, revealing that selection breeding may have a modest nutritional enhancement effect, which has not yet become evident in the G1 generation. This result was agreed with the findings of Zhang et al. [43] regarding the low heritability of most fatty acids in muscle. This demonstrates that, compared to the significantly improved amino acid composition observed in G1 female crabs as mentioned above, genetic enhancement of key fatty acids may require longer breeding cycles or different selection strategies. Considering that whole edible tissues were used for fatty acid relative content analysis in this study, the effect of selective breeding on specific tissues remains to be further explored. Overall, from a fatty acid nutritional perspective, the value of microbial agents lies in their ability to optimize the n-3/n-6 ratio, a key health indicator, while chilled fish proves more effective in directly enhancing core functional fatty acids (EPA and DHA). Although breeding efforts did not exhibit significant negative effects, their potential for enhancing high-value fatty acids in the G1 generation remains underdeveloped. Consequently, achieving the dual objectives of improving the health index and functional enrichment of fatty acid relative content may necessitate the deep integration of breeding programs, specific microbial agents, and lipid-enriched feed strategies in future research.

As well as organic nutrients, the way minerals are deposited also shows the complex effects of breeding, selection and diet on the physiological metabolism of E. sinensis. This is directly related to whether the crab is safe to eat and how nutritious it is. Macronutrients represented by K, Ca, Na, etc. play critical roles in maintaining bone health and cardiovascular function, while trace elements, including Fe, Zn, Cu, etc. are indispensable for physiological processes such as immune regulation and oxygen transport [44]. This study reveals significant tissue- and sex-specific variations in the mineral composition of E. sinensis, with selective breeding and dietary exerting distinct regulatory effects on specific key elements. Regarding macro-minerals, Na and K—closely associated with osmotic regulation—exhibited marked differences across tissues and groups [45]. The highest Na concentration (CF: 13,559.35 ± 1032.96 mg/kg) was observed in the male gonad, whereas the highest K content was found in male muscle tissue (SF: 7400.89 ± 388.25 mg/kg). Of greater significance is the metabolic balance between Ca and P. Ca and P are key minerals participated in various physiological processes in crustaceans and play indispensable roles in life activities. Ca is essential for shell calcification during moulting, while P is integral for exoskeleton formation [46]. In this study, the Ca and P contents in the female gonads of the four experimental groups showed opposite trends, suggesting that Ca and P follow a certain balance in the gonad of female E. sinensis [31,47]. This may relate to the prioritization of specific mineral requirements during gonadal development. Mg is essential for growth, enzyme activation, cellular metabolism and cell division, and it supports ovarian development. In female gonad, Mg concentrations were highest in the NF group and lowest in the SF group, possibly indicating that the ovary in the SF group was fully developed by this time [48]. Fe, Mn, Zn, Cu and Se constitute essential trace elements for human physiological functions. Among them, Fe functions as a critical micronutrient involved in hematopoiesis, oxygen transport, and fluid homeostasis. Zn serves as a vital enzymatic cofactor in nucleic acid and protein biosynthesis. Cu facilitates hemoglobin formation, while Mn supports neurological functions in the central nervous system [49]. Se plays a key role in immunomodulation, redox homeostasis, and genomic stability [50]. In the female gonad, the NF group exhibited the highest levels of Zn and Cu, which was consistent with the previously mentioned Mg levels in the same group. Increased Zn and Cu concentrations during ovarian maturation may reflect metabolic adjustments related to reproduction or the transfer of minerals to the ovary [48]. The highest Fe content was observed in the muscle tissue of both female and male crabs in the SF group. Furthermore, Se was detected in all four experimental groups, with the CF group showing the highest Se levels in female muscle. This indicates that feeding chilled fish effectively promoted Se deposition in female crab muscle. In summary, the mineral composition of E. sinensis is not simply governed by environmental or dietary factors but rather results from the combined effects of genetic background, nutritional levels, and tissue physiological functions. The breeding process, whilst enhancing growth traits, may have altered the efficiency of mineral distribution between gonads and muscle tissue. Meanwhile, chilled fish demonstrate particular value in the enrichment of key trace elements such as Se. These findings provide a novel scientific rationale for optimizing the nutritional quality of minerals in E. sinensis through combining nutritional and genetic selection strategies.

This study demonstrates that the nutritional quality of E. sinensis is governed by complex interactions among genetic selection, feed composition, and sex-related factors. Breeding effects are more pronounced in females, while chilled fish and compound feed systems exhibit distinct advantages in regulating various nutrients. Limitations exist, however, as the study lacked an experimental group feeding the selected G1 strain with chilled fish. Consequently, the maximum potential for high-quality natural feed to enhance the nutritional quality of the selected strain could not be precisely quantified, failing to fully reveal potential synergistic effects between genetics and nutrition. Future research should incorporate this comparative group to comprehensively evaluate the performance ceiling of the selected strain under different nutritional strategies. This will provide more complete scientific evidence for developing precise feeding strategies tailored to superior breeds.

5. Conclusions

This study employed a comprehensive nutritional profiling approach to evaluate the treatment-specific impacts on the quality of E. sinensis across four experimental groups (NF, SF, SF8, CF), analyzing proximate composition, amino acid profiles, fatty acid relative content, and mineral element concentrations. The results indicated that E. sinensis in the chilled fish feeding (CF) group exhibited higher nutritional value and superior performance in key nutritional indicators compared to other groups. Comparative nutritional analysis revealed no statistically significant differences between the SF and SF8 groups, suggesting their primary distinction may reside in their influence on aquatic environmental modulation rather than in the nutrient composition of crab tissues. Future studies should incorporate long-term dynamic monitoring of water quality to further elucidate the relationship between environmental factors and nutritional quality. Regarding the effectiveness of selective breeding, although nutritional improvement was observed in the selected groups, significant gender differences were evident. Females showed more pronounced nutritional improvement, whereas males did not exhibit significant enhancement. In summary, the G1 generation of selected E. sinensis demonstrated superiority over the non-selected group in several nutritional indicators, however, a notable gap remained when compared to the chilled fish feeding group. This study not only clarified the effects of different feeding and breeding strategies on the nutritional quality of E. sinensis but also pointed out directions for optimizing breeding protocols, improving feed formulations and regulating culture environments. These findings offer an important theoretical foundation and practical reference for promoting efficient and sustainable aquaculture practices for E. sinensis.