Controlled Multi-Stage Evaluation of Growth and Physiochemical Traits Between Low- and Normal-Temperature Strains of Scylla paramamosain

Abstract

The mud crab Scylla paramamosain is a key economic crab species along the southern coastal regions of China. This study systematically compared the physiological and biochemical characteristics of low-temperature (LT) and normal-temperature (NT) strains of S. paramamosain at different life stages (juveniles and adults), integrating temperature gradient experiments with conventional aquaculture evaluations. The experimental results revealed the following: (1) Growth superiority: LT-strain crabs exhibited significantly greater final weight, survival rate, hepatopancreatic index, and gonadal index than their NT counterparts (p < 0.05). Moreover, LT individuals displayed an enhanced nutritional profile, with 16.56% higher muscle crude fat and a 23.80% increase in ovarian ash content. (2) Immune competence: Juvenile LT crabs exhibited greater antioxidant capacity at 18–21 °C, with significantly higher total antioxidant capacity (T-AOC) and superoxide dismutase (SOD) activity than NT crabs (p < 0.05). In adults, immune enzyme activity remained superior, particularly in serum acid phosphatase (ACP). (3) Nutritional advantage: LT mature females exhibited higher accumulation of essential amino acids (e.g., lysine, threonine) and polyunsaturated fatty acids (C18:2n-6, C20:2n-6) in the hepatopancreas and gonads (p < 0.05). These findings confirm the LT strain’s superior cold resilience and aquaculture potential, offering practical insights for S. paramamosain selective breeding programs and sustainable aquaculture development.

1. Introduction

The mud crab Scylla paramamosain is well known for its rapid growth, broad salinity tolerance, and large body size, making it one of the leading economic crab species along the southern coastal regions of China [1,2]. Its appealing taste and high nutritional value have made it a popular choice among consumers. According to the China Fishery Statistical Yearbook (2024) [3], aquaculture production of S. paramamosain reached 157,000 tons, yet it remains insufficient to meet the growing market demand. A major factor contributing to this gap is the shortage of high-quality artificial seed stock. Since the 21st century, significant advancements have been made in addressing the challenges posed by wild seed stock, such as the development of pond-based ecological seedling cultivation with a survival rate of up to 77% [4], hybrid breeding that enhances growth rates by 30% [5], and desalination acclimation for selecting crabs with low salinity tolerance [6]. These efforts have significantly improved stock quality and disease resistance, advancing the technology of fully artificial seed production. Simultaneously, S. paramamosain aquaculture has expanded into the lower-temperature inland saline–alkali regions [7] and northern coastal areas [8]. However, a major challenge persists in these regions: the continued shortage of high-quality seed stock suitable for low-temperature cultivation.

As a poikilothermic species, the reproduction, development, and growth of S. paramamosain are highly sensitive to temperature fluctuations [9]. Similarly, sudden temperature shifts in the shrimp Rhynchocinetes durbanensis and the hermit crab Calcinus laevimanus often lead to reduced daily activity, decreased feeding, and metabolic disturbances [10]. When environmental temperatures fall below 25.7 °C, Scylla serrata larvae experience delayed molting and prolonged growth and development [11,12], accompanied by reduced metamorphosis and survival rates [13,14]. Furthermore, when water temperatures drop below 11.7 °C, the growth of S. paramamosain is halted, potentially leading to widespread mortality [15,16]. Disease resistance in crustaceans is closely linked to their immune and antioxidant systems [17,18], both of which are highly susceptible to environmental factors such as salinity, pH, ammonia nitrogen, and temperature [19,20]. Notably, temperature fluctuations particularly affect immune function and antioxidant capacity [21]. Ding et al. [22] demonstrated that low temperatures reduce total hemocyte count (THC) and inhibit immune enzyme activities, including lysozyme (LSZ), in S. serrata. Similarly, Kong et al. [23] showed that low temperatures significantly reduce the activity of antioxidant enzymes, such as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPX) in S. paramamosain, increasing susceptibility to diseases and mortality. In practical aquaculture, annual cold waves and late spring frosts cause substantial economic losses to S. paramamosain culture in China [24], while other economically important marine species, such as Fenneropenaeus chinensis [25], Pseudosciaena crocea [26], and Chlamys nobili [27], which are also vulnerable to low temperatures, have undergone selective breeding for cold tolerance with initial success. In summary, based on its broad salinity tolerance, it is imperative to develop new low-temperature-resistant strains of S. paramamosain to mitigate the impacts of cold weather events on aquaculture.

A collaborative research initiative between the Ningbo Academy of Oceanology and Fishery (Ningbo, China) and Ningbo Huada Haichang Aquatic Technology Co., Ltd. (Ningbo, China), was launched to develop a cold-tolerant strain of S. paramamosain. After several years of selective breeding, a strain with superior traits was developed. Studies on C. nobilis suggest that cold-adapted aquatic organisms accumulate greater nutritional reserves before overwintering to withstand low temperatures [27]. Thus, can the low-temperature strain of S. paramamosain demonstrate superior nutritional value? This study examines the low-temperature strain of S. paramamosain, selected over several years, with the normal-temperature strain as a control. It compares the growth, antioxidant and immune performance, and nutritional quality between the two strains, aiming to provide both theoretical and practical insights for the breeding, improvement, and promotion of low-temperature S. paramamosain strains.

2. Materials and Methods

2.1. Experimental Design

The experimental animal in this study is Scylla paramamosain, which belongs to invertebrates and is a kind of edible crab. In China, ethical approval is generally not mandated for experiments on invertebrates such as crabs under current national guidelines. Between April and May 2023, 32 identical culture tanks (50 cm × 70 cm × 50 cm) were placed in a temperature-controlled greenhouse at Ningbo Huada Haichang Aquatic Technology Co., Ltd. Each tank had a 15 cm square escape-proof plastic mesh at the outlet, and the bottom was covered with 0.8 cm of clean fine sand, offering a substrate for juvenile crabs to burrow and hide. Based on preliminary temperature trials, the temperature gradient experiment was conducted in early April using juvenile crabs from both low-temperature and normal-temperature strains of the same batch (Phase I). Four temperature settings were established: 24 °C, 21 °C, 18 °C, and 15 °C. For each strain and temperature, four replicates were conducted, with 50 healthy juvenile crabs (active, with intact limbs and no signs of disease) introduced into each tank. Before the experiment, crabs were acclimated in the tanks for one day with water at a height of 20 cm; seawater conditions were maintained with a pH of 8.4 ± 0.2, ammonia nitrogen < 0.2 mg/L, nitrite < 0.1 mg/L, dissolved oxygen between 5 and 8 mg/L, temperature of 26 ± 1 °C, and salinity of 22 ± 3‰. Subsequently, the temperature was gradually reduced by 0.5 °C per hour until the target conditions were reached. The experiment began once the target temperature was achieved and remained constant throughout the trial. During the experiment, feed was provided twice daily, at approximately 6:00 and 17:00, with the daily feeding amount constituting 1–4% of the crabs’ body weight. The feeding ratio was consistent across all groups, with adjustments made based on leftover feed. Every three days, fresh seawater pre-adjusted to the target temperature was replaced to maintain stable water quality parameters, except for temperature. Daily observations were made at 19:00 regarding the survival and molting of juvenile crabs, and the experiment continued until they reached stage III.

After the low-temperature stress experiment, juvenile crabs from both the low-temperature and normal-temperature strains were transferred to the intermediate culture phase at the Huada Haichang flat-flow aquaculture pond. The fiberglass flow tanks measured 3 m in length, 2 m in width, and 0.5 m in height, with water salinity maintained at 19 ± 3‰ and pH at 8.2 ± 0.4. The crabs were cultured until they reached an average weight of 41.76 ± 8.72 g. After harvest in mid-June, juvenile crabs from both strains were transferred to experimental ponds at the Huada Haichang aquaculture demonstration site for further cultivation. The experimental ponds spanned 20 acres, with water depths ranging from 1.3 to 1.7 m, pH 8.3 ± 0.4, ammonia nitrogen < 2.0 mg/L, nitrite < 0.1 mg/L, dissolved oxygen between 3 and 5 mg/L, temperature of 26 ± 5 °C, and salinity of 24 ± 5‰. Daily feeding consisted of fresh bivalves and small fish, supplemented with formulated feed. Surveys were conducted monthly from July to October to monitor the growth and conditions of both strains of adult crabs, ensuring their proper development in each pond.

After the cultivation period ended in November, high-quality female crabs from both strains (body weight = 350.88 ± 36.07 g, average carapace length = 8.8 ± 0.65 cm, and average carapace width = 12.45 ± 0.83 cm) were transferred to the Huada Haichang temperature-controlled crab dormitory (dimensions: 36 cm × 26 cm × 27 cm) for overwintering and fattening. During this period, the crabs were fed live razor clams (Sinonovacula constricta), and water changes were performed weekly to meet hydration requirements. During the conditioning period (from February to early March), seawater conditions were maintained with a pH of 8.4 ± 0.2, ammonia nitrogen < 0.2 mg/L, nitrite < 0.1 mg/L, dissolved oxygen between 5 and 8 mg/L, temperature of 21 ± 1 °C, and salinity of 24 ± 3‰. In March 2024, the growth performance and nutritional indicators of these crabs were assessed and compared.

2.2. Sample Collection

After completing the temperature gradient experiment, 20 stage III juvenile crabs from each group were randomly sampled to assess their growth performance. Prior to weighing, surface moisture was removed with filter paper, and the crabs were weighed precisely using an electronic balance (accuracy: 0.001 g). The crabs were then placed in 1.5 mL centrifuge tubes and stored at −20 °C. From July to October 2023, 20 adult crabs from each strain were randomly collected monthly from the Ninghai Huada Haichang aquaculture pond for growth assessment. Prior to weighing, the crabs’ surface moisture was gently wiped off with a dry towel, and their weight was measured accurately using an electronic balance (accuracy: 0.01 g). After weighing, the crabs were promptly transported to the laboratory and anesthetized on ice. Hemolymph (1.0 mL) was extracted using a sterile 1.0 mL syringe from the base of the third walking leg and stored in 1.5 mL centrifuge tubes at −20 °C. The cephalothorax was then separated from the body, and the hepatopancreas, ovaries, and muscle tissues were dissected, weighed precisely, and stored at −20 °C. The remaining tissues and body parts were stored separately in self-sealing bags at −20 °C for future use. The same experimental procedures were applied to the fattened female adult crabs.

2.3. Indicator Measurement

The weight gain rate (WGR (%)), special weight gain rate (SGR (%/d)), survival rate (%), molting rate (%), hepatosomatic index (HSI (%)), ovaryosomatic index (GSI (%)), meat yield (MY (%)) and total edible yield (TEY (%)) were calculated using the following formula [28,29,30]:

$$\mathrm{WGR} (\%)={\displaystyle \frac{(\mathrm{W}1-\mathrm{W}0)}{\mathrm{W}1}}\times 100\%$$

$$\mathrm{SGR} (\%/\mathrm{d})={\displaystyle \frac{(\ln \mathrm{W}1-\ln \mathrm{W}0)}{t}}\times 100\%$$

Survival rate (%) = Ns /No × 100%

Molting rate (%) = Nm /No × 100%

HSI (%) = W_H/W × 100%

GSI (%) = W_G/W × 100%

MY (%) = W_M/W × 100%

TEY (%) = HSI (%) + GSI (%) + MY (%)

where W0 is the average body mass (g) of sampled juvenile crabs in each group of stage I at the beginning of the experiment; W1 is the average body mass (g) of sampled juvenile crabs in each group at the end of the experiment; t is the incubation time; No is the quantity of juvenile crabs in each group at the beginning of the experiment; Ns and Nm are the number of surviving juvenile crabs and the number of juvenile crabs molting to stage III in each group at the end of the experiment, respectively; W_H, W_G and W_M are hepatopancreas mass, ovary mass and muscle mass of adult crabs, respectively; and W is the body mass of adult crabs.

Following the method described by Zhao et al. [31,32], tissue homogenates, hepatopancreas homogenates, and serum were prepared. Antioxidant enzymes, including superoxide dismutase (SOD), total antioxidant capacity (T-AOC), and malondialdehyde (MDA), were measured using a reagent kit produced by Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Additionally, non-specific immune enzymes such as acid phosphatase (ACP) and alkaline phosphatase (ALP) were also assessed.

2.4. Biochemical Analysis

The routine nutritional composition analysis was performed as follows: hepatopancreas, muscle, and ovary samples were weighed and placed in a refrigerator at −80 °C for precooling for 4 h. Subsequently, the samples were placed in a vacuum freeze-drying oven, and the vacuum in the drying chamber was controlled to 20 Pa and −45 °C for 48 h. After freeze-drying, some samples were stored at −80 °C for biochemical analysis, and the rest were dried in a 105 °C drying oven until a constant weight to calculate the dry weight content for amino acid and fatty acid quantification [33]. Crude protein content in these tissues was determined using the Kjeldahl method, and ash content was quantified using the 550 °C ashing method [33]. Crude fat content in the hepatopancreas, muscle, and ovaries was extracted and determined using a chloroform/methanol solution (V/V = 2:1), following the procedure outlined by Folch et al. [34].

Fatty acid composition was analyzed using gas chromatography with an external standard method, following the detection standard JY/T003-1996 [35]. After determining the crude fat content in the hepatopancreas, muscle, and ovaries of adult crabs using a chloroform/methanol solution (V/V = 2:1), fatty acid methyl esters (FAMEs) were extracted with a methanol/sulfuric acid solution, and 0.01% butylated hydroxytoluene (BHT) was added as the antioxidant. Methyl tricosanoate (C23:0; Sigma-Aldrich, Shanghai, China) was used as the internal standard at a concentration of 1.0 mg/mL. Fatty acid profiles were analyzed using gas chromatography–mass spectrometry (GC-MS, Agilent Technologies 7890B-5977A, Santa Clara, CA, USA). Standard curves were constructed by plotting the peak areas of known fatty acid standards, with peak area on the vertical axis and concentration on the horizontal axis. The absolute content of fatty acids in the samples was determined by correlating peak areas with the standard curve [35].

Amino acid analysis was conducted using ion-exchange chromatography for separation and detection. Protein samples were hydrolyzed with 6 mol/L hydrochloric acid (HCl) at 110 °C for 24 h. Afterward, the hydrochloric acid was removed using nitrogen gas, and the samples were re-dissolved in 0.1 mol/L hydrochloric acid loading buffer. The samples were filtered through a 0.22 µm PES ultrafiltration membrane. Finally, the amino acid composition and concentration were determined using an amino acid analyzer (L-8900, Hitachi, Tokyo, Japan) [36].

2.5. Data Processing

Statistical analyses were conducted using SPSS 26 (version 26.0, SPSS Inc., Chicago, IL, USA). Normality was assessed using the Shapiro–Wilk test (α = 0.05), and variance homogeneity was evaluated with Levene’s test (α = 0.05) before parametric analyses. For data following a normal distribution with equal variances (p > 0.05 in both tests), independent-sample t-tests were conducted to compare mean growth parameters, enzyme activity, and nutritional quality metrics in juvenile crabs. Non-normally distributed data were analyzed using Mann–Whitney U tests, while Welch’s t-test was applied for unequal variances. Statistical significance was set at p < 0.05. Figures were generated using OriginPro 2021 (Version 2021, OriginLab Corporation, Northampton, MA, USA), and all data were presented as mean ± standard deviation ($\overline{\mathrm{x}}$ ± SD).

3. Results

3.1. Comparison of Growth Performance Between Low-Temperature and Normal-Temperature Strains of S. paramamosain

3.1.1. The Growth Performance of Juvenile S. paramamosain from Low-Temperature and Normal-Temperature Strains Was Compared Under Different Temperature Conditions

Table 1. Comparison of growth performance of young crabs from low-temperature and normal-temperature strains of S. paramamosain (n = 30).

Item	24 °C		21 °C		18 °C		15 °C
	Low	Normal	Low	Normal	Low	Normal	Low	Normal
Initial body weight/mg	20.81 ± 2.12	19.58 ± 2.47	20.81 ± 2.12	19.58 ± 2.47	20.81 ± 2.12	19.58 ± 2.47	20.81 ± 2.12	19.58 ± 2.47
Final body weight/mg	64.48 ± 2.04 b	55.55 ± 2.36 a	55.51 ± 2.25	55.24 ± 5.01	62.80 ± 2.83 b	54.25 ± 2.88 a	44.90 ± 2.21 b	40.14 ± 1.43 a
WGR/%	209.88 ± 9.78 b	195.62 ± 9.66 a	166.74 ± 10.82 a	182.14 ± 25.59 b	201.79 ± 13.60 b	177.09 ± 14.70 a	115.79 ± 10.61 b	105.01 ± 7.31 a
SGR/(%/d)	7.54 ± 0.21 b	7.22 ± 0.22 a	6.54 ± 0.27	6.90 ± 0.62	3.68 ± 0.15 b	3.39 ± 0.18 a	2.56 ± 0.17 b	2.39 ± 0.12 a
Survival rate/%	71.11 ± 6.94 b	61.11 ± 5.09 a	78.89 ± 8.39 b	60.00 ± 13.33 a	63.33 ± 3.33 b	46.67 ± 8.82 a	63.33 ± 13.33 b	55.56 ± 6.94 a
Molting rate/%	56.67 ± 12.02	57.78 ± 5.09	54.44 ± 5.09 b	44.44 ± 12.62 a	42.22 ± 1.92 b	38.89 ± 1.92 a	0.00	0.00
Period (CI-III) /d	10 to 15	11 to 15	12 to 15	14 to 17	13 to 30	18 to 30	30	30

Note: Data are mean $\overline{\mathrm{x}}$ ± SD. Values in the same row with different superscripts are significantly different (p < 0.05).

presents the growth data of juvenile crabs from both strains at different temperatures. The low-temperature strain exhibited a significantly higher survival rate than the normal-temperature strain at all experimental temperatures (p < 0.05). Except at 21 °C, the low-temperature strain had significantly greater final body weight, weight gain rate, and specific growth rate than the normal-temperature strain (p < 0.05). At 21 °C and 18 °C, the low-temperature strain had a significantly higher molting rate than the normal-temperature strain (p < 0.05). At 15 °C, neither strain exhibited molting.

3.1.2. Comparison of Growth Performance Between Low-Temperature and Normal-Temperature Strains of Adult S. paramamosain

Table 2. Comparison of growth performance of low-temperature and normal-temperature strains of S. paramamosain (n = 20).

Item	Low Temperature	Normal Temperature
Initial body weight (6)/g	41.76 ± 8.70	41.43 ± 8.72
Final body weight (10)/g	345.43 ± 47.11 b	303.13 ± 41.01 a
WGR (6–10)/%	753.01 ± 40.69 b	631.03 ± 48.51 a
SGR (6–10)/(%/d)	1.79 ± 0.04 b	1.66 ± 0.05 a
HSI/%	7.83 ± 0.66 b	6.62 ± 0.94 a
GSI/%	3.09 ± 0.22	3.51 ± 1.01
MY/%	24.15 ± 1.38 b	19.94 ± 4.77 a
TEY/%	35.07 ± 1.24	30.40 ± 5.32

Note: Data are mean $\overline{\mathrm{x}}$ ± SD. Values in the same row with different superscripts are significantly different (p < 0.05).

presents the growth performance of adult S. paramamosain from both the low-temperature and normal-temperature strains. Over the growth period, the low-temperature strain showed significantly higher final body weight, weight gain rate, specific growth rate, hepatopancreas index, and meat yield than the normal-temperature strain (p < 0.05). No significant differences were found between the two strains in ovary index and edible yield (p > 0.05).

Figure 1 illustrates the growth variations of adult S. paramamosain from low-temperature and normal-temperature strains between June and October. From August to October, the average body weight of the low-temperature strain was significantly higher than that of the normal-temperature strain (p < 0.05). In addition, except for the June–July and September–October periods, the weight gain rate and specific growth rate of the low-temperature strain were consistently higher than those of the normal-temperature strain (p < 0.05).

3.1.3. Comparison of Growth Performance in Female Mature S. paramamosain Between Low-Temperature and Normal-Temperature Strains

Table 3. Comparison of nutritional components of low-temperature and normal-temperature strain of S. paramamosain (n = 10).

Item	Low Temperature	Normal Temperature
Body weight/g	422.04 ± 54.46 b	390.26 ± 56.34 a
HSI/%	6.12 ± 1.31	6.15 ± 1.57
GSI/%	18.80 ± 3.71 b	15.01 ± 1.17 a
MY/%	21.27 ± 2.39	20.81 ± 2.61
TEY/%	46.69 ± 3.67 b	42.01 ± 2.43 a

Note: Data are mean $\overline{\mathrm{x}}$ ± SD. Values in the same row with different superscripts are significantly different (p < 0.05).

presents the growth performance of adult crabs reaching ovarian maturity under identical environmental conditions for both low-temperature and normal-temperature strains. The low-temperature strain exhibited significantly higher body mass, ovary index, and edible yield than the normal-temperature strain (p < 0.05). No significant differences were observed between the two strains in hepatopancreas index and meat yield (p > 0.05).

3.2. Comparison of Enzyme Activity Between Low-Temperature and Normal-Temperature Strains of S. paramamosain

3.2.1. Comparison of Antioxidant and Immune Enzyme Activity in Juvenile S. paramamosain from Low-Temperature and Normal-Temperature Strains at Different Temperatures

Figure 2 shows the antioxidant enzyme activity of juvenile S. paramamosain from low-temperature and normal-temperature strains at various temperatures. At 21 °C and 18 °C, the low-temperature strain exhibited significantly higher T-AOC and SOD activity than the normal-temperature strain (p < 0.05). Conversely, at 24 °C and 15 °C, the normal-temperature strain showed significantly higher T-AOC and SOD activity, along with higher MDA content, than the low-temperature strain (p < 0.05).

Figure 3 shows the non-specific immune enzyme activity of juvenile S. paramamosain from low-temperature and normal-temperature strains at different temperatures. At 24 °C, the normal-temperature strain exhibited significantly higher ACP and ALP activity than the low-temperature strain (p < 0.05). At 18 °C, the low-temperature strain showed significantly higher ACP and ALP activity than the normal-temperature strain (p < 0.05). At 21 °C, no significant differences in ACP and ALP activity were observed between the two strains (p > 0.05).

3.2.2. Comparison of Antioxidant and Immune Enzyme Activity in Adult S. paramamosain Between Low-Temperature and Normal-Temperature Strains

Figure 4 presents the antioxidant enzyme activity in the hepatopancreas of adult S. paramamosain from both low-temperature and normal-temperature strains. In August, T-AOC enzyme activity in the hepatopancreas of the normal-temperature strain was significantly higher than that of the low-temperature strain (p < 0.05). No significant differences in T-AOC enzyme activity were observed between the two strains in the remaining months (p > 0.05). From July to October, SOD enzyme activity in the hepatopancreas of the low-temperature strain was consistently higher than that in the normal-temperature strain (p < 0.05). Conversely, MDA content in the normal-temperature strain was significantly higher than in the low-temperature strain (p < 0.05).

Figure 5 illustrates the antioxidant enzyme activity in the serum of adult S. paramamosain from both low-temperature and normal-temperature strains. From August to October, T-AOC activity in the serum of the low-temperature strain was significantly higher than that of the normal-temperature strain (p < 0.05). From August to September, SOD enzyme activity in the serum of the low-temperature strain was significantly higher than in the normal-temperature strain (p < 0.05). Throughout the rearing period, MDA content in the serum of the normal-temperature strain remained consistently higher than that of the low-temperature strain (p < 0.05).

Figure 6 shows the non-specific immune enzyme activity in the hepatopancreas of adult S. paramamosain from both low-temperature and normal-temperature strains. From July to October, ACP enzyme activity in the hepatopancreas of the low-temperature strain was significantly higher than that of the normal-temperature strain (p < 0.05). In August and September, ALP enzyme activity in the hepatopancreas of the low-temperature strain was significantly higher than in the normal-temperature strain (p < 0.05). However, in July and October, ALP enzyme activity in the hepatopancreas of the normal-temperature strain was significantly higher than that of the low-temperature strain (p < 0.05).

Figure 7 presents the non-specific immune enzyme activity in the serum of adult S. paramamosain from both low-temperature and normal-temperature strains. Throughout the rearing period, except in October, ALP and ACP activities in the serum of the low-temperature strain were significantly higher than those in the normal-temperature strain (p < 0.05).

3.3. Comparison of Conventional Biochemical Composition in Mature Female Mud Crabs Between Low-Temperature and Normal-Temperature Strains

Table 4. Proximate biochemical composition in hepatopancreas, ovary, and muscle of mature female mud crabs between low-temperature and normal-temperature strains (% wet weight, n = 10).

Item	Low Temperature	Normal Temperature
Hepatopancreas moisture content	52.11 ± 2.19	53.89 ± 3.38
Crude protein/hepatopancreas wet weight	41.47 ± 1.26	39.08 ± 1.11
Total fat/hepatopancreatic wet weight	61.04 ± 0.95 a	65.60 ± 0.35 b
Hepatopancreatic ash content	3.57 ± 0.12	3.42 ± 0.03
Muscle moisture content	74.08 ± 4.18	73.95 ± 5.25
Crude protein/muscle wet weight	80.58 ± 1.66	79.25 ± 0.85
Total fat/muscle wet weight	7.04 ± 0.43 b	6.04 ± 0.14 a
Muscle ash content	5.86 ± 0.17	5.99 ± 0.10
Ovarian moisture content	47.08 ± 3.64	45.85 ± 4.84
Crude protein/ovarian wet weight	62.42 ± 4.02	59.90 ± 3.58
Total fat/ovarian wet weight	21.70 ± 1.13 b	18.85 ± 1.19 a
Ovarian ash content	5.15 ± 0.36 b	4.16 ± 0.06 a

Note: Data are mean $\overline{\mathrm{x}}$ ± SD. Values in the same row with different superscripts are significantly different (p < 0.05).

summarizes the conventional biochemical composition of the hepatopancreas, muscle, and ovary in adult S. paramamosain from low-temperature and normal-temperature strains. The low-temperature strain had significantly higher crude fat content in both the muscle and ovary, as well as higher ash content in the ovary, compared to the normal-temperature strain (p < 0.05). In contrast, the crude fat content in the hepatopancreas of the low-temperature strain was significantly lower than that in the normal-temperature strain (p < 0.05). No significant differences were found in other parameters (p > 0.05); however, the low-temperature strain showed higher levels of crude protein and ash in the hepatopancreas, and higher moisture and crude protein content in the muscle and ovary, compared to the normal-temperature strain.

3.4. Comparison of Nutritional Composition in Mature Female Mud Crabs Between Low-Temperature and Normal-Temperature Strains

3.4.1. Comparison of Fatty Acid Composition in the Hepatopancreas, Muscle, and Ovary of Mature Female Mud Crabs Between Low-Temperature and Normal-Temperature Strains

Table 5. Comparison of fatty acid composition in the hepatopancreas of mature female mud crabs between low-temperature and normal-temperature strains (mg/g dry weight, n = 3).

Item	Low Temperature	Normal Temperature
C12:0	0.19 ± 0.02 b	0.15 ± 0.01 a
C14:0	4.39 ± 0.36 b	3.34 ± 0.33 a
C16:0	32.29 ± 1.70	26.08 ± 6.20
C18:0	11.91 ± 0.37	10.83 ± 0.73
C20:0	0.77 ± 0.08 a	0.91 ± 0.02 b
∑SFA	49.55 ± 1.72	41.31 ± 6.73
C16:1n-7	21.69 ± 1.81	17.44 ± 2.61
C18:1n-9	35.18 ± 3.61	38.91 ± 1.86
C20:1n-9	4.99 ± 0.19	3.84 ± 0.72
C22:1n-9	0.92 ± 0.03	0.73 ± 0.15
∑MUFA	62.79 ± 1.88	60.93 ± 4.98
C18:2n-6	6.10 ± 0.44 b	2.98 ± 0.38 a
C18:3n-3	3.42 ± 0.18 b	2.62 ± 0.44 a
C18:4n-3	2.62 ± 0.15 b	1.59 ± 0.39 a
C20:2n-6	8.32 ± 1.10 b	5.49 ± 0.74 a
C20:4n-3	1.93 ± 0.18 b	1.05 ± 0.21 a
C20:4n-6	2.34 ± 0.17 b	1.09 ± 0.05 a
C20:5n-3	15.11 ± 0.42 b	9.21 ± 1.30 a
C22:5n-3	2.21 ± 0.28 b	1.54 ± 0.14 a
C22:6n-3	20.78 ± 4.80	13.25 ± 0.26
∑PUFA	59.80 ± 4.21 b	41.96 ± 3.37 a
total	172.14 ± 4.64 b	144.19 ± 13.58 a
DHA/EPA	9.34 ± 1.04 b	8.68 ± 0.76 a
∑n-3PUFA	46.07 ± 5.35 b	29.26 ± 2.25 a
∑n-6PUFA	16.84 ± 0.68 b	9.58 ± 0.39 a

Note: Data are mean $\overline{\mathrm{x}}$ ± SD. Values in the same row with different superscripts are significantly different. ∑SFA, total saturated fatty acid; ∑MUFA, total mono-unsaturated fatty acid; ∑PUFA, total polyunsaturated fatty acid. Fatty acids with no <0.10 mg total fatty acids are shown in the table.

shows that the fatty acid composition of the hepatopancreas in both strains of mature female mud crabs was identical, with 19 fatty acids detected, including 5 saturated fatty acids (SFAs), 4 monounsaturated fatty acids (MUFAs), and 10 polyunsaturated fatty acids (PUFAs). The predominant fatty acids were C16:0, C16:1n-7, C18:0, C18:1n-9, C20:5n-3, and C22:6n-3. These six major fatty acids accounted for 80.26% and 79.57% of the total fatty acids in the hepatopancreas of the low-temperature and normal-temperature strains, respectively. In the low-temperature strain, the levels of C12:0, C14:0, C18:2n-6, C18:3n-3, C18:4n-3, C20:2n-6, C20:4n-3, C20:4n-6, C20:5n-3, C22:5n-3, ∑PUFA, total, DHA/EPA, ∑n-3PUFA, and ∑n-6PUFA were significantly higher than those in the normal-temperature strain (p < 0.05), while the level of C20:0 was significantly lower (p < 0.05).

Table 6. Comparison of fatty acid composition in the muscle of mature female mud crabs between low-temperature and normal-temperature strains (mg/g dry weight, n = 3).

Item	Low Temperature	Normal Temperature
C16:0	2.80 ± 0.09	2.39 ± 0.34
C18:0	1.97 ± 0.09 b	1.76 ± 0.09 a
∑SFA	5.05 ± 0.12	4.32 ± 0.44
C16:1n-7	1.56 ± 0.23 b	0.99 ± 0.10 a
C18:1n-9	3.94 ± 0.61	3.82 ± 0.95
C20:1n-9	0.24 ± 0.05 b	0.13 ± 0.03 a
∑MUFA	5.78 ± 0.58	4.98 ± 0.90
C18:2n-6	0.98 ± 0.29 b	0.28 ± 0.05 a
C18:3n-3	0.13 ± 0.03	0.18 ± 0.01
C20:2n-6	0.49 ± 0.02 b	0.25 ± 0.06 a
C20:4n-6	0.55 ± 0.00	0.63 ± 0.14
C20:5n-3	3.93 ± 0.66	3.35 ± 0.10
C22:5n-3	0.26 ± 0.05 b	0.15 ± 0.03 a
C22:6n-3	2.61 ± 0.18	3.25 ± 0.38
∑PUFA	9.05 ± 0.83	13.16 ± 0.81
total	19.87 ± 1.15	13.28 ± 0.83
DHA/EPA	10.01 ± 1.20 a	21.46 ± 4.46 b
∑n-3PUFA	7.02 ± 0.80	7.01 ± 0.27
∑n-6PUFA	2.02 ± 0.29 b	1.17 ± 0.21 a

Note: Data are mean $\overline{\mathrm{x}}$ ± SD. Values in the same row with different superscripts are significantly different. ∑SFA, total saturated fatty acid; ∑MUFA, total mono-unsaturated fatty acid; ∑PUFA, total polyunsaturated fatty acid. Fatty acids with no <0.10 mg total fatty acids are shown in the table.

shows that the fatty acid composition of the muscle was identical in both strains, with 18 fatty acids detected, including 5 saturated fatty acids, 4 monounsaturated fatty acids, and 9 polyunsaturated fatty acids. The major fatty acids were C16:0, C16:1n-7, C18:0, C18:1n-9, C20:5n-3, and C22:6n-3, which accounted for 80.84% and 82.29% of the total muscle fatty acids in the low-temperature and normal-temperature strains, respectively. In the low-temperature strain, the levels of C16:1n-7, C18:0, C18:2n-6, C20:1n-9, C20:2n-6, C22:5n-3, and ∑n-6PUFA were significantly higher than those in the normal-temperature strain (p < 0.05), whereas the DHA/EPA ratio was significantly lower (p < 0.05).

Table 7. Comparison of fatty acid composition in the ovary of mature female mud crabs between low-temperature and normal-temperature strains (mg/g dry weight, n = 3).

Item	Low Temperature	Normal Temperature
C12:0	0.15 ± 0.01	0.16 ± 0.01
C14:0	2.61 ± 0.08	3.19 ± 0.41
C16:0	31.01 ± 2.69	30.28 ± 1.77
C18:0	11.64 ± 0.85	11.64 ± 0.17
C20:0	0.56 ± 0.07	0.51 ± 0.02
∑SFA	45.94 ± 1.99	45.78 ± 2.32
C16:1n-7	27.14 ± 4.25	20.43 ± 2.38
C18:1n-9	38.97 ± 5.27	38.21 ± 7.57
C20:1n-9	2.39 ± 0.48	1.88 ± 0.17
C22:1n-9	0.61 ± 0.02 a	0.70 ± 0.04 b
∑MUFA	69.11 ± 3.82	61.22 ± 5.35
C18:2n-6	9.07 ± 1.76 b	1.73 ± 0.32 a
C18:3n-3	1.97 ± 0.32	1.66 ± 0.22
C18:4n-3	0.47 ± 0.06 a	0.70 ± 0.05 b
C20:2n-6	3.71 ± 0.46 b	2.16 ± 0.14 a
C20:4n-3	0.58 ± 0.05 a	0.84 ± 0.06 b
C20:4n-6	2.24 ± 0.27 a	3.22 ± 0.53 b
C20:5n-3	16.10 ± 2.60 a	17.81 ± 0.67 b
C22:5n-3	2.82 ± 0.48 b	2.10 ± 0.16 a
C22:6n-3	14.57 ± 0.50 a	17.74 ± 3.30 b
∑PUFA	51.57 ± 1.99 b	48.02 ± 3.58 a
total	166.65 ± 4.32 b	155.02 ± 7.84 a
DHA/EPA	5.28 ± 1.08 a	8.42 ± 1.13 b
∑n-3PUFA	36.51 ± 1.38 a	40.85 ± 3.70 b
∑n-6PUFA	15.06 ± 1.65 b	7.16 ± 0.89 a

Note: Data are mean $\overline{\mathrm{x}}$ ± SD. Values in the same row with different superscripts are significantly different. ∑SFA, total saturated fatty acid; ∑MUFA, total mono-unsaturated fatty acid; ∑PUFA, total polyunsaturated fatty acid. Fatty acids with no <0.10 mg total fatty acids are shown in the table.

shows that the fatty acid composition of the ovary was identical in both strains, with 19 fatty acids detected, including 5 saturated fatty acids, 4 monounsaturated fatty acids, and 10 polyunsaturated fatty acids. The predominant fatty acids were C16:0, C16:1n-7, C18:0, C18:1n-9, C20:5n-3, and C22:6n-3, which accounted for 83.57% and 88.31% of the total fatty acids in the ovary of the low-temperature and normal-temperature strains, respectively. In the low-temperature strain, the levels of C18:2n-6, C20:2n-6, C22:5n-3, PUFA, total, and ∑n-6PUFA were significantly higher than those in the normal-temperature strain (p < 0.05). However, the levels of C18:4n-3, C20:4n-3, C20:4n-6, C20:5n-3, C22:1n-9, C22:6n-3, DHA/EPA, and ∑n-3PUFA were significantly lower in the low-temperature strain (p < 0.05).

3.4.2. Comparison of Amino Acid Composition in the Hepatopancreas, Muscle, and Ovary of Female Mature Mud Crabs from Low-Temperature and Normal-Temperature Strains

Table 8. Comparison of amino acid composition in the hepatopancreas of female mature mud crabs from low-temperature and normal-temperature strains (mg/g dry weight, n = 3).

Item	Low Temperature	Normal Temperature
Threonine (Thr)	14.80 ± 1.59	11.02 ± 1.81
Valine (Val)	16.02 ± 1.40 b	11.41 ± 1.64 a
Methionine (Met)	4.64 ± 1.19	3.10 ± 0.42
Isoleucine (Ile)	13.83 ± 1.01 b	9.47 ± 1.88 a
Leucine (Leu)	20.67 ± 4.50	16.67 ± 2.65
Phenylalanine (Phe)	12.78 ± 2.72	10.43 ± 1.43
Lysine (Lys)	22.64 ± 1.65 b	16.20 ± 3.20 a
∑EAA	109.70 ± 9.62 b	85.80 ± 0.75 a
Aspartic acid (Asp)	32.21 ± 2.86 b	22.23 ± 4.04 a
Serine (Ser)	10.67 ± 1.66	8.70 ± 0.76
Glutamine (Glu)	37.69 ± 7.31	28.96 ± 4.82
Glycine (Gly)	14.58 ± 2.62	11.03 ± 1.18
Alanine (Ala)	14.78 ± 2.05 b	10.56 ± 1.24 a
Cysteine (Cys)	3.34 ± 0.68	2.70 ± 0.12
Tyrosine (Tyr)	15.19 ± 1.17 b	11.72 ± 0.01 a
Histidine (His)	10.08 ± 0.83 b	7.06 ± 1.17 a
Arginine (Arg)	18.42 ± 3.50	14.29 ± 2.69
Proline (Pro)	12.76 ± 1.93	10.70 ± 0.82
∑NEAA	169.73 ± 21.56	127.95 ± 15.99
TAA	275.11 ± 33.71	206.25 ± 28.99
EAA/TAA	0.38 ± 0.00	0.38 ± 0.01
EAA/NEAA	0.62 ± 0.01	0.61 ± 0.03
∑DAA	127.24 ± 16.09 b	94.92 ± 12.23 a
DAA/TAA	0.46 ± 0.01	0.46 ± 0.01

Note: Data are mean $\overline{\mathrm{x}}$ ± SD. Values in the same row with different superscripts are significantly different. Amino acids with <1 mg of total essential amino acids are not listed in table.

,

Table 9. Comparison of amino acid composition in the muscle of female mature mud crabs from low-temperature and normal-temperature strains (mg/g dry weight, n = 3).

Item	Low Temperature	Normal Temperature
Threonine (Thr)	26.10 ± 2.72	25.50 ± 2.02
Valine (Val)	26.04 ± 2.93	25.17 ± 1.69
Methionine (Met)	11.20 ± 0.33	10.05 ± 1.25
Isoleucine (Ile)	23.70 ± 3.21	23.27 ± 2.01
Leucine (Leu)	42.98 ± 4.87	42.05 ± 3.48
Phenylalanine (Phe)	24.38 ± 3.20	23.40 ± 1.87
Lysine (Lys)	45.53 ± 5.02	44.45 ± 3.21
∑EAA	199.93 ± 21.82	193.89 ± 15.16
Aspartic acid (Asp)	55.76 ± 6.96	53.96 ± 3.29
Serine (Ser)	21.98 ± 2.47	21.46 ± 1.67
Glutamine (Glu)	93.36 ± 10.78	90.57 ± 7.43
Glycine (Gly)	33.68 ± 3.68	31.59 ± 4.75
Alanine (Ala)	40.28 ± 7.05	31.60 ± 3.03
Cysteine (Cys)	4.84 ± 0.00	5.05 ± 0.78
Tyrosine (Tyr)	22.72 ± 3.14	20.94 ± 1.78
Histidine (His)	14.12 ± 2.26	13.08 ± 0.91
Arginine (Arg)	53.51 ± 5.14	55.11 ± 5.31
Proline (Pro)	24.20 ± 2.33	24.16 ± 1.67
∑NEAA	364.44 ± 35.82	347.51 ± 26.54
TAA	564.37 ± 56.56	541.40 ± 41.62
EAA/TAA	0.35 ± 0.01	0.36 ± 0.00
EAA/NEAA	0.55 ± 0.02	0.56 ± 0.01
∑DAA	270.18 ± 27.39	252.05 ± 18.47
DAA/TAA	0.48 ± 0.01 b	0.47 ± 0.00 a

Note: Data are mean $\overline{\mathrm{x}}$ ± SD. Values in the same row with different superscripts are significantly different. Amino acids with <1 mg of total essential amino acids are not listed in table.

and

Table 10. Comparison of amino acid composition in the ovary of female mature mud crabs from low-temperature and normal-temperature strains (mg/g dry weight, n = 3).

Item	Low Temperature	Normal Temperature
Threonine (Thr)	26.78 ± 0.27	26.88 ± 0.34
Valine (Val)	30.58 ± 0.33	30.48 ± 0.55
Methionine (Met)	14.02 ± 0.05	14.40 ± 0.98
Isoleucine (Ile)	23.89 ± 0.14	23.81 ± 0.34
Leucine (Leu)	42.26 ± 0.26	42.22 ± 1.08
Phenylalanine (Phe)	23.29 ± 0.99	22.89 ± 0.66
Lysine (Lys)	32.40 ± 0.97	32.14 ± 0.99
∑EAA	193.22 ± 2.14	192.83 ± 4.70
Aspartic acid (Asp)	42.40 ± 1.41	42.04 ± 0.64
Serine (Ser)	29.92 ± 0.16	29.98 ± 1.03
Glutamine (Glu)	70.42 ± 1.08	70.62 ± 1.46
Glycine (Gly)	19.25 ± 0.27	19.21 ± 0.31
Alanine (Ala)	23.32 ± 1.35	22.54 ± 0.44
Cysteine (Cys)	5.02 ± 0.01	5.06 ± 0.27
Tyrosine (Tyr)	24.23 ± 0.95	24.85 ± 0.63
Histidine (His)	15.90 ± 0.87	15.54 ± 0.31
Arginine (Arg)	35.20 ± 0.36	35.39 ± 1.44
Proline (Pro)	29.57 ± 0.86	29.55 ± 0.42
∑NEAA	295.23 ± 4.33	294.79 ± 4.33
TAA	488.45 ± 6.47	487.62 ± 8.98
EAA/TAA	0.40 ± 0.00	0.40 ± 0.00
EAA/NEAA	0.65 ± 0.00	0.65 ± 0.01
∑DAA	202.91 ± 5.59	202.16 ± 3.75
DAA/TAA	0.42 ± 0.01	0.41 ± 0.00

Note: Data are mean $\overline{\mathrm{x}}$ ± SD. Amino acids with <1 mg of total essential amino acids are not listed in table.

show that the amino acid composition in the hepatopancreas, muscle, and ovary of mature female mud crabs was identical in both low-temperature and normal-temperature strains. Nine essential amino acids (EAAs) and eight non-essential amino acids (NEAAs) were detected, totaling 17 amino acids. The levels of valine (Val), isoleucine (Ile), lysine (Lys), aspartic acid (Asp), alanine (Ala), tyrosine (Tyr), and histidine (His) in the hepatopancreas of the low-temperature strain were significantly higher than those in the normal-temperature strain (p < 0.05), with both essential amino acids (EAAs) and umami amino acids (DAAs) showing significant increases (p < 0.05). Although the amino acid content in the muscle and ovary of the low-temperature strain was higher than that in the normal-temperature strain, the differences were not significant (p > 0.05). However, the ratio of umami amino acids (DAAs) to total amino acids (TAAs) in the low-temperature strain was significantly higher than in the normal-temperature strain (p < 0.05).

4. Discussion

4.1. Growth Performance and Biochemical Differences Between Low-Temperature and Normal-Temperature Strains of Juvenile and Female Mature Mud Crabs

Temperature is a key factor affecting the growth, development, and survival of crustaceans [37,38]. In the temperature stress experiments, the low-temperature strain exhibited the highest final body weight, weight gain rate, specific growth rate, and survival rate at all tested temperatures. At 18 °C and 21 °C, the molting rate of juvenile crabs from the low-temperature strain was the highest, indicating superior adaptation to temperature variations and enhanced growth performance. These findings align with studies by Miao et al. [26] on cold-tolerant L. crocea and Zhang et al. [27] on cold-resistant C. nobilis. Between 18 °C and 24 °C, the molting rate of juvenile crabs in both strains decreased as the temperature lowered, and developmental time increased. This suggests that lower environmental temperatures lead to prolonged molting and developmental periods in fiddler crab juveniles [11,12], while metamorphosis and survival rates decrease [13,14]. At 15 °C, neither strain of juvenile crabs reached the III stage, further indicating that excessively low temperatures can cause growth stagnation or death [16]. Therefore, temperature settings for stress experiments with juvenile mud crabs should be carefully considered, with a recommended minimum of 15 °C. Subsequent aquaculture and fattening experiments showed that the low-temperature strain exhibited the highest final body weight and the greatest amounts of crude fat and ash in the muscle and ovary of mature adult female crabs. This further highlights the superior growth performance of the low-temperature strain during the adult fattening stage.

The hepatopancreas and ovaries are essential organs for lipid metabolism and nutrient storage in crustaceans, providing energy for growth and metabolism [39,40]. The indices of the hepatopancreas and ovaries reflect the accumulation of nutrients in these organs [41]. In this study, the low-temperature strain exhibited the highest hepatopancreas and ovary indices, indicating its greater capacity for nutrient accumulation and enhanced feeding and growth abilities under long-term cold conditions. In addition to the hepatopancreas index, meat yield and edible ratio are important indicators of the culinary value of mud crabs [42]. Both meat yield and edible ratio were highest in the low-temperature strain, suggesting that this strain offers higher nutritional value.

4.2. Differences in Antioxidant and Immune Performance Between Low-Temperature and Normal-Temperature Strains of Juvenile and Adult Mud Crabs

Superoxide dismutase (SOD) is a key antioxidant enzyme in crustaceans [43], playing a vital role in antioxidant defense, immune response, and cellular metabolism. Low SOD activity leads to lipid oxidation, generating malondialdehyde (MDA) and other lipid peroxides, which are toxic to cells. Higher concentrations cause more damage [44]. Total antioxidant capacity (T-AOC) is a key indicator of an organism’s overall antioxidant ability [45]. At temperatures between 18 °C and 24 °C, juvenile crabs from the low-temperature strain exhibited higher SOD and T-AOC activities and lower MDA levels. At 15 °C, juvenile crabs from the normal-temperature strain exhibited the highest SOD and T-AOC activities, suggest an activated antioxidant defense against temperature-induced reactive oxygen species (ROS). However, the simultaneous rise in MDA suggests that oxidative damage still occurred despite these compensatory mechanisms, likely due to an imbalance between ROS generation and clearance. This phenomenon is consistent with findings in S. serrata under cold stress, where increased antioxidant activity was insufficient to counteract lipid peroxidation [46]. The heightened response in the normal-temperature strain may indicate a lack of genetic adaptation to prolonged cold exposure, resulting in metabolic inefficiency and aggravated oxidative stress. These findings suggest that juvenile crabs from the low-temperature strain have superior antioxidant capacity in colder environments, making them less prone to oxidative damage. The superior antioxidant capacity of low-temperature-strain juveniles may stem from epigenetic regulation of SOD and CAT genes.

In crustaceans’ immune defense system, two important non-specific phosphohydrolases, acid phosphatase (ACP) and alkaline phosphatase (ALP), catalyze the hydrolysis of phosphoesters and transfer phosphate groups [47,48]. This enhances nutrient transport and forms a hydrolytic enzyme system to eliminate foreign invaders. These enzymes are essential for crustacean survival and growth. This study showed that at 24 °C, juvenile crabs from the normal-temperature strain exhibited the highest ACP and ALP activities, while enzyme activity in the low-temperature strain did not differ significantly. However, at 18 °C, the trend reversed, suggesting that the low-temperature strain exhibits superior immune performance only under cooler conditions [25]. Throughout the cultivation period, adult crabs from the low-temperature strain consistently exhibited the lowest MDA levels in both the hepatopancreas and serum, along with the highest ACP and ALP activities. These findings align with the juvenile temperature gradient experiment results, further highlighting that adult crabs from the low-temperature strain have superior antioxidant capacity and enhanced immune performance during cultivation.

The superior antioxidant and immune performance of the low-temperature strain observed in this study suggests that it may modulate oxidative stress responses via the Nrf2-Keap1 signaling pathway [49] or enhance innate immunity through the Toll-like receptor pathway [50]. Future studies should use qPCR to profile key gene expression within these pathways and apply CRISPR/Cas9 gene-editing technology [51] to validate gene function. The obtained phenotypic variation data provide essential parameters for establishing breeding indices based on trait association analysis, particularly for aquaculture in the Bohai Bay region, where the average annual water temperature is ≤18 °C. This will further support the selection and genetic improvement of cold-tolerant S. paramamosain strains.

4.3. Nutritional Composition Differences Between Low-Temperature and Normal-Temperature Strains of Female Mature Mud Crabs

The fatty acid content and composition in the hepatopancreas, muscle, and ovary of mud crabs are important indicators of their breeding value [52]. This study shows that the fatty acid composition in the hepatopancreas, muscle, and ovary of adult crabs from both strains is identical, with the main fatty acids being C16:0, C16:1n-7, C18:0, C18:1n-9, C20:5n-3, and C22:6n-3. Among these, C16:0, C16:1n-7, C18:0, and C18:1n-9 are energy-storing fatty acids accumulated during ovarian development [53], providing metabolic energy for post-embryonic development. Their content does not significantly differ between the hepatopancreas and ovary, in agreement with previous reports [35].

C18:2n-3 (LA), C18:3n-3 (LNA), C20:5n-3 (EPA), and C22:6n-3 (DHA) are essential fatty acids (EFAs) that mud crabs cannot synthesize and must obtain from food. These fatty acids are crucial for egg incubation, juvenile growth and development, molting, metamorphosis, and maintaining metabolic functions in adult crabs [54]. In this study, the low-temperature strain had significantly higher levels of LA in the hepatopancreas, muscle, and ovary than the normal-temperature strain. This may explain the superior growth, molting, metamorphosis, and metabolic performance of the low-temperature strain. Additionally, the low-temperature strain had significantly higher total polyunsaturated fatty acid (∑PUFA) levels in both the hepatopancreas and ovary, with ∑n-3PUFA levels in the hepatopancreas increasing by about 60%. These findings are consistent with studies on cold-tolerant strains of C. nobilis [27]. ∑n-3PUFAs, known for their beneficial effects on cardiovascular health, neural development, and immune function [55], have gained significant attention. This suggests that, in addition to its growth performance, the low-temperature strain offers enhanced nutritional value, better meeting consumer demand.

Organisms’ amino acids include essential amino acids (EAAs) and non-essential amino acids (NEAAs). Ten amino acids, including Thr, Ile, and Val, are essential for the growth and development of mud crabs, while six amino acids, including Phe, Asp, and Glu, contribute to the flavor and taste of the food [56]. Research by Li et al. [57] indicates that cold-adapted aquatic species exhibit elevated amino acid concentrations. These compounds function as cryoprotectants, stabilizing cellular structures during low-temperature stress while enhancing metabolic flexibility via protein turnover. This adaptive mechanism mitigates the effects of suppressed enzymatic activity under cold conditions. In this study, the low-temperature strain of mud crabs had slightly higher levels of EAAs, NEAAs, total amino acids (TAAs), and delicious amino acids (DAAs) in the hepatopancreas, muscle, and ovary than the normal-temperature strain. This suggests that the edible parts of the low-temperature strain have superior nutritional value and flavor, in agreement with findings for Eriocheir sinensis [58] and Paralithodes camtschaticus [59].

In the hepatopancreas, the low-temperature strain had significantly higher levels of Val, Ile, Lys, and His than the normal-temperature strain. Elevated levels of these amino acids enhance growth and development [60,61], antioxidant capacity [62,63], and nutritional value [64] in crustaceans. These findings suggest that adult mud crabs from the low-temperature strain have higher nutritional value and superior growth performance and antioxidant capacity compared to the normal-temperature strain.

5. Conclusions

The findings indicate that the low-temperature strain of S. paramamosain possesses significant advantages over its normal-temperature counterpart. In the juvenile stage, it exhibits superior cold adaptability, whereas in adulthood, it surpasses the normal-temperature strain in growth rate, immune function, and antioxidant capacity, with a significantly improved nutritional profile. Based on a comprehensive comparative analysis, we recommend prioritizing the large-scale cultivation of this selectively bred strain in northern coastal regions. Additionally, implementing a gradual cooling acclimation protocol (1 °C/day reduction) during the juvenile stage could further enhance cold tolerance, addressing the increasing consumer demand for high-quality S. paramamosain. Future research should aim to elucidate the molecular mechanisms of its cold adaptation and the regulatory pathways governing nutrient accumulation, thereby offering theoretical insights and practical guidance for the selection, enhancement, and commercialization of cold-tolerant aquaculture species.