Investigation of Plasticity in Morphology, Organ Traits and Nutritional Composition in Chinese Soft-Shelled Turtle (Pelodiscus sinensis) Under Different Culturing Modes

Abstract

The Chinese soft-shelled turtle (Pelodiscus sinensis) is an aquatic reptile prized for its nutritional and health benefits. Given its adaptability to various culturing modes including the greenhouse, pond and rice culturing modes, we conducted a comparative analysis of the morphology, organ trait and nutritional composition of turtles cultured in three culturing modes. This study investigated the plasticity of morphology and physiology, as well as the variations in nutritional composition across varying culturing modes. The results demonstrated that after approximately 120 days of cultivation, significant changes were observed in the morphology, physiology and nutritional composition of turtles from each culturing mode. In terms of morphology, rice turtles exhibited an arched shell shape, broad plastron, elongated limbs, narrow interocular distance and slender head and neck. Pond turtles displayed similar morphological characteristics to rice turtles, with the additional features of a flattened body shape and narrower plastron. Greenhouse turtles presented a flattened shell shape, narrow plastron, shortened limbs, wider interocular distance and stocky head and neck. Regarding the organ characteristics, the specific weights of liver, viscera, internal fat lumps and condition factors were significantly higher in greenhouse turtles compared to rice turtles and pond turtles (p < 0.05). Conversely, the specific weights of the back carapace, calipash and edible part were significantly lower than those in rice turtles and pond turtles (p < 0.05). Nutritional analysis revealed that crude protein, total amino acid, essential amino acid, flavor amino acid, pharmacodynamic amino acid, collagen and EPA+DHA contents were significantly higher in rice turtles and pond turtles than greenhouse turtles (p < 0.05). However, crude fat and unsaturated fatty acid contents were significantly higher in greenhouse turtles than in rice turtles and pond turtles (p < 0.05). In summary, Chinese soft-shelled turtles exhibited significant morphological and organ plasticity in response to different culturing modes. While the rice and pond culturing modes could enhance the nutritional quality of turtles to some extent, the impact of commercial feed on fatty acid profiles must be carefully considered.

1. Introduction

Environmental diversity and biotic interactions can induce adaptive modifications in phenotypes, facilitating the development of biodiversity [1,2]. Long-term adaptive modifications resulting in the formation of heritable phenotypes are referred to as adaptive evolution [3]. Conversely, environmental plasticity refers to the short-term adaptive modifications of phenotypes in response to an environment [4]. Owing to anthropogenic activities, climate change and other influences, the rate of phenotypic adaptation in organisms has significantly accelerated [5,6]. In agricultural production, specific habitat conditions and nutrient supplies are designed to achieve optimal production outcomes. This intentional manipulation of the environment accelerates biological adaptation and promotes phenotypic plasticity in agricultural organisms. Over successive generations, organisms in agricultural organisms develop specific phenotypes desired by humans. However, research on adaptive changes in organisms within agricultural production is limited, hindering a deeper understanding of organismal plasticity, generational evolution in variety breeding and biological welfare in agricultural production.

The Chinese soft-shelled turtle (Pelodiscus sinensis) belongs to Reptilia, Testudoformes, Trionychidae and Pelodiscus and is a secondary aquatic reptile. In the wild, it inhabits freshwater ecosystems such as rivers, lakes and reservoirs. It plays a significant role in the aquaculture industry of East and Southeast Asia due to its edible and medicinal value [7,8]. Unlike other aquatic animals, the Chinese soft-shelled turtle exhibits amphibious characteristics, allowing it to adapt to diverse culturing modes. These culturing modes can be roughly categorized into captive and ecological culturing modes [9,10,11]. The greenhouse culturing mode is a typical model of captive culturing modes [9]. Owing to the biological characteristics of a prolonged growth cycle and ectothermy, the effective growth period of the Chinese soft-shelled turtle is confined to May through October annually, with the remainder of year being characterized by hibernation. To extend the effective growth period, an intensive greenhouse culturing mode is widely adopted in China. This mode enhances growth rates and the yield of turtles through the provision of abundant commercial feed and high-density cultivation under optimal temperature conditions. Simultaneously, this mode also mitigates the limitations imposed by hibernation and ectothermy in turtles [12]. Nevertheless, the drawbacks of water quality, increased frequency of disease outbreaks, reduced activity space and the diminished quality associated with the greenhouse culturing mode are detrimental to the health, welfare and food safety of turtles [13,14]. The representative ecological culturing modes are the pond culturing mode and rice culturing mode [10,11,15]. In accordance with the ecological habits of the Chinese soft-shelled turtle, farmers utilize ponds and rice fields as habitats for turtles. By adopting low-density cultivation and moderate feeding practices, they ensure that Chinese soft-shelled turtles enjoy ample living space, superior water quality and adequate sunlight exposure [16]. These conditions are not present in the greenhouse culturing mode. While ecological culturing modes may decrease the unit yield, they are advantageous for enhancing the nutritional quality, market price and animal welfare of Chinese soft-shelled turtles [17].

To balance the advantages and limitations of both types of modes, known as the “two-stage” mode, two types of culturing modes are often employed in relay. In this relay mode, the greenhouse culturing mode is employed in the initial year for high-density cultivation to enhance growth rate and unit yield. In the subsequent appropriate season of the following year, juvenile turtles from the greenhouse culturing mode are transferred to ponds or rice fields, where ecological culturing modes are used to enhance quality. This relay culturing mode not only shortens the culture cycle and enhances yield but also improves the quality of turtles cultivated in greenhouses. However, during the transition between culturing modes, turtles are confronted with alterations in living conditions, including habitat, environment and food supply, which may trigger adaptive responses. There are few studies on the adaptive changes during the transition between different culturing modes. Based on the above reasons, juvenile Chinese soft-shelled turtles from a greenhouse culturing mode were selected as the subject. Culturing experiments were conducted sequentially in three different culturing modes: the greenhouse culturing mode, pond culturing mode and rice culturing mode. Comprehensive comparative analyses of morphology, biological indicators and nutritional composition were conducted to evaluate the impact of culturing mode practices on phenotypic plasticity and the enhancement of nutritional quality. This study aims to provide valuable reference to the ecological adaptability and nutritional improvement of Chinese soft-shelled turtles.

2. Materials and Methods

2.1. Chinese Soft-Shelled Turtle for Experimentation and Culturing Experiment Design

The Chinese soft-shelled turtles utilized in this study were the Japanese strain of Chinese soft-shelled turtle, cultivated in the greenhouse of Zhejiang Fisheries Technical Extension Center after hatching in July 2022. Following one year of cultivation in the greenhouse (May 2023), juvenile female turtles, with a body weight ranging from 300 g to 350 g (329.15 ± 19.31 g), were selected for this culturing experiment.

The rice culturing experiment (R group, hereafter referred to as rice-turtle) was executed at the rice/fishery integrated cultured experimental base of Zhejiang University in Jiaxing, Zhejiang Province, China (120°26′26″ E, 30°32′08″ N). The rice field covered an area of 100 m², with a row spacing of 20 cm × 30 cm and a water depth of 0.3 m. Two weeks after rice planting, 25 juvenile turtles were cultured at a stocking density of 0.25 individuals/m², and three parallel experimental groups were established. The experiment lasted 120 days, from 2 June to 29 September 2023.

The pond culturing experiment (P group, hereafter referred to as pond-turtle) was executed at Zhejiang Fisheries Technical Extension Center (120°32′13″ E, 30°22′33″ N). Each pond covered an area of 600 m², a water depth of 1.5 m and sandy sediment. The pond was equipped with anti-escape settings and sunbathing platforms. A total of 300 juvenile turtles were cultured at a stocking density of 0.50 individuals/m², and three parallel experimental groups were established. The experiment lasted 121 days, from 29 May to 26 September 2023.

The greenhouse culturing experiment (G group, hereafter referred to as greenhouse-turtle) was also executed at Zhejiang Fisheries Technical Extension Center. The cement pool situated in the dark greenhouse devoid of natural light covered an area of 30 m², with a water depth of 1.2 m and hardened cement base. Each pool was stocked with 300 juvenile turtles at a stocking density of 10 individuals/m², and three parallel experimental groups were established. The experiment lasted 120 days, from 26 May to 23 September 2023.

Throughout the culturing experiment, the experimental groups were fed a special commercial feed (Haihuang, Hangzhou, China, containing 43% crude protein and 8% crude fat) for Chinese soft-shelled turtles twice daily at 8:00 am and 3:00 pm, each time in accordance with 3% of body weight. For the rice-turtle group, clean water was not altered but replenished according to the requirements of rice cultivation. In the pond-turtle group, clean water was replaced every 10 days, with each replacement amounting to 1/10 of pond. For the greenhouse-turtle group, clean water was replaced every 3 days with each replacement being 1/3 of pool.

2.2. Measurement and Sampling

Upon completion of the culturing experiment, 30 healthy, uninjured turtles were randomly selected from each culturing experiment (10 individuals were collected from each parallel experimental group of the respective culturing experiment) and transported to the College of Life Sciences, Zhejiang University, for subsequent experiments. The body weights of the turtles in the rice-turtle group ranged from 436.98 g to 643.31 g (477.90 ± 26.78 g), those in the pond-turtle group ranged from 639.40 g to 721.45 g (667.29 ± 22.86 g), and those in the greenhouse-turtle group ranged from 687.39 g to 850.67 g (761.67 ± 49.22 g).

A total of 22 morphological indexes of turtle were determined following ether anesthesia. The morphological indexes included head and neck indexes (proboscis breadth, PRB; proboscis length, PRL; snout length, SL; head breadth, HB; head height, HH; interocular distance, ID; head length, HL; neck length, NL; neck breadth, NB), back indexes (body height, BH; body length, BL; back carapace length, BCL; back carapace breadth, BCB), plastron indexes (plastron length, PL; plastron breadth, PB; plastron concave breadth, PCB; calipash breadth, CB; calipash thickness, CT; tail length, TL; tail breadth, TB), and limbs indexes (fore limb length, FLL; hind limb length, HLL). Body weight (BW) was measured using an electronic scale (accuracy: 0.01 g, Yukovit Electronic Technology Co., LTD., Suzhou, China), while morphological indexes were recorded with a vernier caliper (accuracy: 0.01 mm, ASIMETO, Munich, Germany) and referred to schematic diagram of morphological indexes of Chinese soft-shelled turtle (Figure 1, drawing upon research paper by Qu [18]). The back carapace perimeter (BCP) was measured using Image J software (Version 7.0) following image capture.

After morphological measurements, each turtle was individually weighed and subsequently dissected. A total of 5 organs and tissues, including the viscera, liver, back carapace, calipash and internal fat lump, were sequentially extracted. The remaining body (the body after the removal of all organs, internal fat lump, back carapace and blood) was regarded as the edible part. All organs and tissues were cleaned with purified water, dried with filter paper and then weighed (accuracy: 0.01 g). After weighing, the hind limb muscles and calipash tissues were dissected and combined into six pooled samples, placed in self-sealing bags and stored at −80 °C for nutritional analysis.

2.3. Preprocessing of Morphological and Organ Indexes Data

To eliminate the impact of individual variations on the analysis of morphological and organ indexes, the morphological proportions were normalized using the back carapace length (BCL), obtaining 22 proportional indexes for subsequent analysis. The biological indexes were derived from body weight, and their coefficients were standardized for further analysis. The formula for calculating organ proportions was presented as follows [18,19]:

visceral index (VW/BW, %) = (visceral weight/body weight) × 100%;

liver index (LW/BW, %) = (liver weight/body weight) × 100%;

internal fat lump index (IW/BW, %) = (internal fat lump weight/body weight) × 100%;

back carapace index (BCW/BW, %) = (back carapace weight/body weight) × 100%;

calipash index (CW/BW, %) = (calipash weight/body weight) × 100%;

edible portion index (EPW/BW, %) = [(body weight − visceral weight − back carapace weight)/body weight] × 100%;

condition factor index(a) (BW/BCL³, /) = body weight/(back carapace length)³ × 100;

condition factor index(b) (EPWW/BCL³, /) = (body weight visceral weight − back carapace weight)/(back carapace length)³ × 100.

2.4. Nutritional Index Detection and Calculation

The proximate composition of muscle was determined following the standardized methods of the Association of Official Analytical Chemists (AOAC) [20]. Moisture content was assessed by determining the weight loss after drying the sample at 105 °C for 24 h. Ash content was quantified by incinerating the sample in muffle furnace at 550 °C for 6 h and subsequently measuring the residual weight. The crude fat content was determined using the Soxhlet extraction method and quantified with an automated Soxhlet extraction system (FOSS Soxtec 8000, Hillerød, Denmark). The crude protein content was determined using the Kjeldahl method with Kjeldahl apparatus (FOSS 8400, Hillerød, Denmark). The proximate composition was measured in g/100 g.

The pretreatment for determination of conventional amino acids of muscle and hydroxyproline of calipash were conducted via acid hydrolysis. We accurately weighed both muscle and calipash samples, transferred them into separate hydrolysis tubes, added 4 mL of HCl solution (6 mol/L) and hydrolyzed them at 110 °C for 22 h. The hydrolysate was diluted with ultrapure water and acid removed by nitrogen gas blowdown. The hydrolysate was diluted using HCl solution (0.02 mol/L) and filtered through microporous filter membrane (0.22 μm) to obtain the sample solution for detection. The pretreatment for the determination of tryptophan of muscle was alkaline hydrolysis. We accurately weighed muscle samples and transferred them to hydrolysis tube. Then, 3 mL NaOH solution (4.3 M) was added, followed by the introduction of nitrogen gas. The mixture was then hydrolyzed for 20 h and subsequently transferred to 25 mL centrifuge tube. Following centrifugation (6000 r/min, 5 min) of the hydrolysate, the supernatant was collected and filtered through microporous filter membrane (0.22 μm) to obtain the sample solution for detection. The amino acid solution to be detected was quantified using an automated amino acid analyzer (L-8900, Hitachi, Tokyo, Japan). The contents of amino acids were measured in g/100 g.

We accurately weighed muscle samples and transferred them to 100 mL flask. Subsequently, we added 8 mL of ultrapure water and 10 mL of HCL. The flask was subsequently hydrolyzed in a water bath maintained at 80 °C for a duration of 1 h. Subsequently, ethanol (95%) and diethyl ether were added to the sample to extract the lipid components. Subsequently, 4 mL NaOH–methanol solution (2%) was added and the mixture was heated at 45 °C for 30 min. Following this, 4 mL of a 14% BF₃–methanol solution was introduced and the reaction mixture was again heated at 45 °C for 30 min. Finally, 3 mL hexane was added to the mixture for extraction over a period of 2 min, followed by phase separation. The supernatant was then collected and filtered through microporous filter membrane (0.45 μm) to obtain the solution for detection. Fatty acids were analyzed using gas chromatography–mass spectrometry (Agilent 7890A-5975C, Shanghai, China) via the external standard method (with cholesterol as the external standard). The determined fatty acid content was quantified in g/100 g.

Based on the amino acid scoring pattern model recommended by the Food and Agriculture Organization (FAO) and the World Health Organization (WHO) in 1973 [21], as well as the whole egg protein amino acid model, we calculated the amino acid score (AAS), chemical score (CS), and essential amino acid index (EAAI) to evaluate the nutritional value of amino acids in muscle of Chinese soft-shelled turtle across three different groups.

2.5. Statistical Analysis

Statistical analyses were conducted using SPSS 26.0 software with one-way ANOVA. Following the successful completion of normality and homogeneity of variance tests, Duncan’s multiple comparison test was subsequently utilized to assess the significance of differences between means, with a significance level set at p < 0.05. Results were presented as mean ± standard deviation (SD). Linear discriminant analysis (LDA) was conducted on the morphological proportional data using the MASS and tidyverse packages in R (Version 4.3.3). The discriminant methods based on morphological characteristics for three groups of Chinese soft-shelled turtles were established, and the results were visualized using the ggplot2 package in R. Proportional data of morphology and biology were utilized to construct two matrices and Mantel correlation analysis was carried out using the linkET and dplyr packages in R (Version 4.3.3). The Spearman’s correlation method was selected for the calculations to examine the relationships between morphological and organ indexes. Visualization of the results was achieved using the R package ggplot2.

3. Results

3.1. Morphological Analysis and Discriminant Function

Among the 22 morphological proportion examined, 11 exhibited significant differences between groups (

Table 1. Morphological proportion of P. sinensis from three culture modes (n = 30).

Morphological Proportions	Code	Groups
		R	P	G
Proboscis breadth/back carapace length, PRB/BCL	x1	0.040 ± 0.005	0.038 ± 0.003	0.040 ± 0.006
Proboscis length/back carapace length, PRL/BCL	x2	0.026 ± 0.005	0.030 ± 0.004	0.025 ± 0.004
Snout length/back carapace length, SL/BCL	x3	0.034 ± 0.007	0.039 ± 0.006	0.035 ± 0.008
Head breadth/back carapace length, HB/BCL	x4	0.169 ± 0.005	0.171 ± 0.006	0.178 ± 0.009
Head height/back carapace length, HH/BCL	x5	0.144 ± 0.008	0.146 ± 0.008	0.143 ± 0.008
Interocular distance/back carapace length, ID/BCL	x6	0.031 ± 0.002 a	0.032 ± 0.002 a	0.035 ± 0.002 b
Head length/back carapace length, HL/BCL	x7	0.342 ± 0.022	0.354 ± 0.030	0.343 ± 0.036
Neck length/back carapace length, NL/BCL	x8	0.491 ± 0.055 b	0.474 ± 0.053 a	0.470 ± 0.033 a
Neck breadth/back carapace length, NB/BCL	x9	0.143 ± 0.008 a	0.152 ± 0.011 b	0.151 ± 0.010 b
Body length/back carapace length, BL/BCL	x10	1.860 ± 0.056 b	1.829 ± 0.058 b	1.786 ± 0.055 a
Back carapace breadth/back carapace length, BCB/BCL	x11	0.892 ± 0.015	0.883 ± 0.018	0.881 ± 0.032
Plastron length/back carapace length, PL/BCL	x12	0.785 ± 0.018 a	0.793 ± 0.018 ab	0.804 ± 0.021 b
Plastron breadth/back carapace length, PB/BCL	x13	0.860 ± 0.017 b	0.832 ± 0.034 a	0.827 ± 0.021 a
Plastron concave breadth/back carapace length, PCB/BCL	x14	0.377 ± 0.014 b	0.367 ± 0.014 a	0.369 ± 0.013 a
Calipash breadth/back carapace length, CB/BCL	x15	0.144 ± 0.006 b	0.144 ± 0.008 b	0.137 ± 0.004 a
Calipash thickness/back carapace length, CT/BCL	x16	0.035 ± 0.003	0.035 ± 0.004	0.037 ± 0.004
Tail length/back carapace length, TL/BCL	x17	0.111 ± 0.010	0.097 ± 0.011	0.104 ± 0.012
Tail breadth/back carapace length, TB/BCL	x18	0.123 ± 0.015	0.117 ± 0.012	0.117 ± 0.018
Fore limb length/back carapace length, FLL/BCL	x19	0.487 ± 0.047 b	0.493 ± 0.036 b	0.456 ± 0.024 a
Hind limb length/back carapace length, HLL/BCL	x20	0.577 ± 0.035 b	0.594 ± 0.048 b	0.539 ± 0.042 a
Body height/back carapace length, BH/BCL	x21	0.322 ± 0.019	0.320 ± 0.017	0.316 ± 0.012
Back carapace perimeter/back carapace length, BCP/BCL	x22	3.046 ± 0.189 a	3.094 ± 0.166 a	3.725 ± 0.210 b

R, rice culturing mode; P, pond culturing mode; G, greenhouse culturing mode. n = 30 indicates that the number of statistical samples in each group was 30. Results are presented as the mean ± standard deviation (SD) of 30 replicates (same as in Table 2). The same lines of different superscript letters indicate that there are significant differences between the two groups (p < 0.05).

). Specifically, in the R group, NL/BCL (x8), PB/BCL (x13) and PCB/BCL (x14) were significantly higher compared to the P and G groups (p < 0.05). Additionally, BL/BCL (x10), CB/BCL (x15), FLL/BCL (x19) and HLL/BCL (x20) in both the R and P groups were significantly higher than those in the G group (p < 0.05). Conversely, ID/BCL (x6) and BCP/BCL (x22) were significantly higher in the G group compared to the R and P groups (p < 0.05). Furthermore, NB/BCL (x9) was significantly higher in the G and P groups compared to the R group (p < 0.05). Lastly, PL/BCL (x12) was significantly higher in the G group compared to the R group (p < 0.05).

A linear discriminant function was developed based on 22 morphological proportions to differentiate between populations of Chinese soft-shelled turtles in the three culturing modes. The visualized results demonstrate that the two linear discriminant functions constructed were capable of accurately classifying the morphological indexes of the three groups of turtles in a two-dimensional projection. Specifically, these functions achieved complete separation between groups and relative aggregation within groups (Figure 2). The degree of explanation of linear discriminant function 1 (LD1) in the discriminant classification model is 0.879, while that of linear discriminant function 2 (LD2) is 0.121. For details regarding the classification equations and results of the linear discriminant function, please refer to Table S1 in the supplementary materials.

3.2. Organ Indexes Analysis

Significant inter-group variations were observed in eight organ proportions across the three groups (

Table 2. Organ proportions of P. sinensis from three culture modes (%, n = 30).

Organ Proportions	Groups
	R	P	G
Visceral index, VW/BW	16.079 ± 1.663 a	15.413 ± 1.402 a	17.787 ± 1.023 b
Liver index, LW/BW	3.022 ± 0.451 a	3.686 ± 0.161 b	4.398 ± 0.283 c
Internal fat lump index, IFW/BW	4.246 ± 0.393 a	7.402 ± 0.809 b	6.237 ± 0.469 c
Back carapace index, BCW/BW	15.991 ± 1.197 b	15.906 ± 1.468 b	13.548 ± 0.577 a
Calipash index, CW/BW	6.094 ± 0.553 b	6.051 ± 0.908 b	5.060 ± 0.574 a
Edible portion index, EPW/BW	63.581 ± 1.872 b	62.114 ± 0.560 ab	60.330 ± 4.430 a
Condition factor index(a), BW/BCL3(/)	15.787 ± 0.632 a	15.953 ± 0.873 a	19.176 ± 1.524 b
Condition factor index(b), EPW/BCL3(/)	12.518 ± 0.754 a	12.453 ± 0.481 a	15.345 ± 1.251 b

The same lines of different superscript letters indicate that there are significant differences between the two groups (p < 0.05).

). The proportions of VW/BW, LW/BW, IFW/BW, BW/BCL³ and EPW/BCL³ in Group G were significantly higher compared to those in Groups R and P (p < 0.05). Conversely, the proportions of BCW/BW and CW/BW were significantly higher in Groups R and P than those in Group G (p < 0.05). Additionally, the proportion of EPW/BW was significantly higher in Group R compared to Group G (p < 0.05).

3.3. Correlation Analysis Between Morphological and Biological Characteristics

The correlation analysis revealed a multitude of significant associations between the 22 morphological indexes and the 8 organ indexes. There was a significant positive correlation between LV/BW, IFW/BW, BW/BCL³, EPW/BCL³ and ID/BCL (p < 0.05). Additionally, BCW/BW, EPW/BW and EPW/BCL³ showed significant positive correlations with BCP/BCL (p < 0.05). Furthermore, a significant positive correlation was observed between EPW/BW and HLL/BCL (p < 0.05) (Figure 3).

3.4. Proximate Composition

The proximate composition of muscle across the three groups was largely comparable, with the moisture content being the highest, followed by crude protein, ash and crude fat. However, variations in the concentrations of the same components were observed across the different groups (

Table 3. Proximate composition of muscles of P. sinensis from three culture modes (g/100 g, wet weight, n = 5).

Proximate Compositions	Groups
	R	P	G
Moisture	78.577 ± 0.066	78.817 ± 0.576	79.397 ± 0.254
Ash	0.960 ± 0.036 ab	0.910 ± 0.016 a	1.030 ± 0.033 b
Crude protein	20.450 ± 0.265 b	19.183 ± 0.127 a	18.820 ± 0.221 a
Crude fat	0.713 ± 0.031 a	0.867 ± 0.059 ab	0.957 ± 0.114 b

n = 5 indicates that the number of statistical samples in each group is 5. Results are presented as the mean ± standard deviation (SD) of 5 replicates (same as in Table 4 and Table 5). The same lines of different superscript letters indicate that there are significant differences between the two groups (p < 0.05).

). The crude protein content in Group R was significantly higher than that in Groups G and P (p < 0.05). Additionally, the ash and crude fat contents were highest in Group G compared to the other two groups (p < 0.05). There was no significant difference in moisture between the three groups (p > 0.05).

3.5. Amino Acids

A total of 18 amino acids were detected in the muscles of all three groups (

Table 4. Amino acid indexes of muscles of P. sinensis from three culture modes (g/100 g, wet weight, n = 5).

Tissue	Amino Acids	Groups
		R	P	G
Muscle	Threonine, Thr	0.818 ± 0.005 b	0.809 ± 0.002 b	0.786 ± 0.009 a
	Valine, Val @	0.831 ± 0.012 a	0.829 ± 0.011 a	0.969 ± 0.021 b
	Methionine, Met #	0.518 ± 0.003 c	0.491 ± 0.004 b	0.427 ± 0.011 a
	Isoleucine, Ile @	0.873 ± 0.016 b	0.835 ± 0.007 a	0.841 ± 0.003 a
	Leucine, Leu #,@	1.446 ± 0.016 b	1.434 ± 0.005 b	1.391 ± 0.009 a
	Phenylalanine, Phe #	0.738 ± 0.006	0.741 ± 0.004	0.754 ± 0.005
	Lysine, Lys #	1.732 ± 0.030 c	1.655 ± 0.012 b	1.580 ± 0.027 a
	Tryptophan, Trp	0.157 ± 0.003 c	0.149 ± 0.002 b	0.139 ± 0.001 a
	∑EAAs	7.113 ± 0.075 b	7.083 ± 0.027 a	6.746 ± 0.041 a
	Histidine, His	0.576 ± 0.019 b	0.489 ± 0.012 a	0.745 ± 0.017 c
	Arginine, Arg #	0.982 ± 0.007 b	0.968 ± 0.007 b	0.906 ± 0.009 a
	Serine, Ser	0.733 ± 0.007	0.718 ± 0.005	0.726 ± 0.007
	Proline, Pro	0.706 ± 0.007 c	0.668 ± 0.020 b	0.504 ± 0.006 a
	Cysteine, Cys #	0.368 ± 0.014 c	0.323 ± 0.008 b	0.114 ± 0.011 a
	Tyrosine, Tyr #	0.609 ± 0.012 b	0.589 ± 0.007 b	0.453 ± 0.009 a
	Aspartic acid, Asp #,&	1.605 ± 0.006 b	1.567 ± 0.009 a	1.623 ± 0.015 b
	Glutamic acid, Glu #,&	2.823 ± 0.062 b	2.783 ± 0.027 ab	2.689 ± 0.011 a
	Glycine, Gly #,&	0.856 ± 0.013 a	0.833 ± 0.007 a	0.922 ± 0.014 b
	Alanine, Ala &	1.214 ± 0.014 b	1.156 ± 0.042 b	1.032 ± 0.031 a
	∑NEAAs	10.471 ± 0.077 c	10.094 ± 0.072 b	9.714 ± 0.005 a
	∑TAAs	17.584 ± 0.007 c	17.177 ± 0.071 b	16.460 ± 0.044 a
	∑FAAs &	6.497 ± 0.052 b	6.339 ± 0.075 a	6.266 ± 0.022 a
	∑PAAs &	11.676 ± 0.037 c	11.383 ± 0.039 b	10.858 ± 0.018 a
	∑BCAAs @	3.151 ± 0.036 b	3.239 ± 0.018 c	3.061 ± 0.017 a
	∑EAAs/TAAs (%)	40.453 ± 0.431 a	41.237 ± 0.213 b	40.982 ± 0.141 ab
	∑FAAs/TAAs (%)	38.068 ± 0.151 b	36.948 ± 0.296 a	36.906 ± 0.353 a
	∑PAAs/TAAs (%)	66.402 ± 0.214 b	66.272 ± 0.056 ab	65.968 ± 0.074 a
	∑EAAs/NEAAs (%)	67.943 ± 1.219 a	70.177 ± 0.618 b	69.440 ± 0.404 ab
Calipash	Hydroxyproline, Hyp #	16.149 ± 0.241 c	15.653 ± 0.247 b	14.796 ± 0.171 a

EAAs indicates essential amino acids, NEAAs indicates non-essential amino acids and TAAs indicates total amino acids. FAAs ^& indicates flavor amino acids, marked using “&” in the table. PAAs indicates pharmacodynamic amino acids, marked using “^#” in the table. BCAAs ^@ indicates branched chain amino acids, marked using “@” in the table.

). The total amino acid (TAA) content in the muscles within the R, P and G groups was 17.584 g/100 g, 17.177 g/100 g and 16.460 g/100 g, respectively. The concentrations of the 18 amino acids followed the same trend, with glutamic acid (Glu) exhibiting the highest content, followed by aspartic acid (Asp) and lysine (Lys), while tryptophan (Trp) had the lowest content. Regarding the amino acid composition, the concentrations of eight essential amino acids (EAAs) in Groups R, P and G were 7.113 g/100 g, 7.083 g/100 g and 6.746 g/100 g, respectively. The levels of 10 non-essential amino acids (NEAAs) were 10.471 g/100 g, 10.094 g/100 g and 9.714 g/100 g, respectively. The concentrations of four flavor amino acids (FAAs) were 6.497 g/100 g, 6.339 g/100 g and 6.266 g/100 g, respectively. The amounts of ten pharmacodynamic amino acids (PAAs) were 11.676 g/100 g, 11.383 g/100 g and 10.858 g/100 g, respectively. The concentrations of three branched-chain amino acids (BCAAs) were 3.151 g/100 g, 3.239 g/100 g and 3.061 g/100 g, respectively. The aforementioned five amino acid indexes in the R and P groups were significantly elevated compared to those in the G group (p < 0.05). Furthermore, the hydroxyproline (Hyp) content of the calipash was 16.149 g/100 g, 15.653 g/100 g and 14.796 g/100 g, respectively. The Hyp content was also significantly higher in both Groups R and P compared to Group G (p < 0.05).

The nutritional value of protein is assessed based on the proportion of amino acid categories. The proportions of EAAs/TAAs in muscles of the R, P and G groups were 40.453%, 41.237% and 40.982%, respectively. The proportions of FAAs/TAAs were 38.068%, 36.948% and 36.906%, respectively. The proportions of PAAs/TAAs were 66.402%, 66.272% and 65.968%, respectively. The proportions of EAAs/NEAAs were 67.943%, 70.177% and 69.440%, respectively.

In the amino acid scoring standard (AAS), for Groups R and P, the primary limiting amino acid was valine (Val), followed by threonine (Thr) as the secondary limiting amino acid. For Group G, the primary limiting amino acids were methionine (Met) + cysteine (Cys), with Val being the secondary limiting amino acid. In the chemical scoring standard (CS), for Groups R and P, the primary limiting amino acid was Val, followed by Met + Cys as the secondary limiting amino acids. For Group G, Met + Cys served as the primary limiting amino acids, while phenylalanine (Phe) + tyrosine (Tyr) were the secondary limiting amino acids. The essential amino acid indexes (EAAIs) of the R, P and G groups were 80.417, 84.791 and 78.020, respectively (Table S2).

3.6. Fatty Acids

The comprehensive analysis of muscle fatty acid profiles within the R, P and G groups revealed a total of seventeen fatty acids (

Table 5. Fatty acid indexes of muscles of P. sinensis from three culture modes (g/100 g, wet weight, n = 5).

Fatty Acid	Groups
	R	P	G
Myristic acid, C14:0	1.199 ± 0.005 a	1.780 ± 0.020 c	1.559 ± 0.036 b
Pentadecanoic acid, C15:0	0.743 ± 0.004 b	0.818 ± 0.007 c	0.433 ± 0.004 a
Palmitic acid, C16:0	18.732 ± 0.058 b	21.253 ± 0.143 c	18.112 ± 0.155 a
Heptadecanoic acid, C17:0	0.749 ± 0.010 c	0.397 ± 0.007 b	0.220 ± 0.005 a
Stearic acid, C18:0	5.080 ± 0.030 b	5.929 ± 0.039 c	4.949 ± 0.017 a
Eicosanoic acid, C20:0	0.130 ± 0.002 b	0.125 ± 0.002 a	0.132 ± 0.002 b
ΣSFAs	26.634 ± 0.074 b	30.301 ± 0.160 c	25.404 ± 0.172 a
Palmitoleic acid, C16:1	7.200 ± 0.024 c	6.813 ± 0.061 b	6.521 ± 0.058 a
Heptadecenoic acid, C17:1	0.467 ± 0.007 c	0.362 ± 0.005 b	0.270 ± 0.005 a
Oleic acid, C18:1n-9	29.047 ± 0.235 a	31.083 ± 0.105 c	29.886 ± 0.060 b
Eicosenoic acid, C20:1	7.580 ± 0.028 c	2.963 ± 0.041 a	3.449 ± 0.069 b
Nervonic acid, C24:1	0.700 ± 0.005 a	1.051 ± 0.008 b	1.068 ± 0.006 c
ΣMUFAs	44.993 ± 0.179 c	42.271 ± 0.186 b	41.194 ± 0.089 a
Linoleic acid C18:2n-6 @	8.076 ± 0.056 a	9.812 ± 0.093 b	17.700 ± 0.195 c
Linolenic acid, C18:3n-3 #	2.021 ± 0.021 a	2.800 ± 0.002 b	2.901 ± 0.007 c
Arachidonic acid, C20:4n-6 @	2.637 ± 0.050 c	0.465 ± 0.008 a	0.656 ± 0.010 b
Eicosapentaenoic acid (EPA), C20:5n-3 #	6.388 ± 0.019 b	6.179 ± 0.075 a	6.161 ± 0.042 a
Docosadienoic acid, C22:2	0.755 ± 0.005 c	0.413 ± 0.009 b	0.212 ± 0.012 a
Docosahexaenoic acid (DHA), C22:6n-3 #	7.748 ± 0.022 b	7.884 ± 0.065 c	6.786 ± 0.028 a
ΣPUFAs	27.624 ± 0.119 a	27.553 ± 0.033 a	34.415 ± 0.211 b
ΣUFAs	72.617 ± 0.063 b	69.824 ± 0.160 a	75.608 ± 0.165 c
EPA+DHA	14.135 ± 0.040 b	14.063 ± 0.084 b	12.947 ± 0.045 a
n-3 #	16.156 ± 0.059 b	16.863 ± 0.083 c	15.847 ± 0.038 a
n-6 @	10.713 ± 0.057 b	10.278 ± 0.087 a	18.356 ± 0.203 c
n-6/n-3 (/)	0.663 ± 0.001 b	0.610 ± 0.008 a	1.158 ± 0.012 c
UFAs/SFAs (/)	2.727 ± 0.010 b	2.304 ± 0.017 a	2.976 ± 0.026 c
PUFAs/SFAs (/)	1.037 ± 0.002 b	0.909 ± 0.004 a	1.355 ± 0.017 c

SFAs indicates saturated fatty acids. MUFAs indicates monounsaturated fatty acids. PUFAs indicates polyunsaturated fatty acids. UFAs indicates unsaturated fatty acids. n-3 indicates omega-3 polyunsaturated fatty acids, marked using “^#” in the table. n-6 indicates omega-6 polyunsaturated fatty acids, marked using “^@” in the table.

), comprising six saturated fatty acids (SFAs), five monounsaturated fatty acids (MUFAs) and six polyunsaturated fatty acids (PUFAs). The SFA contents in the muscle within the R, P and G groups were 26.634 g/100 g, 30.301 g/100 g and 25.404 g/100 g, respectively. The MUFA contents were 44.993 g/100 g, 42.271 g/100 g and 41.194 g/100 g, respectively. Both the SFA and MUFA contents were significantly higher in the P and R groups compared to the G group (p < 0.05). The PUFA contents in the muscle within the R, P and G groups were 27.624 g/100 g, 27.553 g/100 g and 34.415 g/100 g, respectively. The unsaturated fatty acid (UFA) contents were 72.617 g/100 g, 69.824 g/100 g and 75.608 g/100 g, respectively. Both the PUFA and UFA contents were significantly higher in the G group compared to the P and R groups (p < 0.05).

Regarding the types and composition of UFAs, the contents of eicosapentaenoic acid (EPA) + docosahexaenoic acid (DHA) in Groups R, P and G were 14.135 g/100 g, 14.063 g/100 g and 12.947 g/100 g, respectively. The omega-3 family fatty acid (n-3) contents were 16.156 g/100 g, 16.863 g/100 g and 15.847 g/100 g, respectively. The above fatty acid indexes of Groups P and R were significantly higher than those of Group G (p < 0.05). The omega-6 family fatty acid (n-6) contents were 10.713 g/100 g, 10.278 g/100 g and 18.356 g/100 g, respectively. The n-6/n-3 ratios were 0.663, 0.610 and 1.158, respectively. The UFA/SFA ratios were 2.727, 2.304 and 2.976, respectively. The PUFA/SFA ratios were 1.037, 0.909 and 1.355, respectively. The above fatty acid indexes of Group G were significantly higher than those of Groups P and R (p < 0.05).

4. Discussion

4.1. Plasticity of Culturing Modes on Morphology

The alterations of culturing mode can be regarded as anthropogenic modifications of the environment, potentially accelerating adaptive alterations in farmed animals [22]. In this study, significant differences were observed in 11 out of 22 morphological proportions of Chinese soft-shelled turtles across different groups. Further analysis using linear discriminant analysis on the morphological proportions revealed no overlap between the groups, suggesting that the morphological plasticity of Chinese soft-shelled turtles had undergone rapid divergent alterations in response to different culturing modes. In this study, we categorized the culturing mode as a single variable and initially determined the changes in the morphological proportions of Chinese soft-shelled turtles under different culturing modes using linear discriminant analysis (a supervised classification model) and one-way ANOVA. This method represented a valid analytical approach. However, it was inherently limited by the constraints of one-way analysis and linear discriminant analysis, which precluded it from being considered a perfect technique. In analogous experiments, it is recommended that researchers explore alternative mathematical analysis techniques. For instance, they could employ a diverse array of machine learning algorithms to construct a robust discriminant analysis framework and concurrently, utilize one-way ANOVA analysis, Bonferroni correction and False Discovery Rate (FDR) methods to mitigate the risk of Type I errors.

Environmental changes can induce morphological adaptations in animals, enabling them to better suit their specific living conditions. This process is often regarded as the plastic adaptation of animals in response to their environment [23,24]. The PL/BCL of the R group was smaller than that of the G group, whereas the PB/BCL and PCB/BCL were larger in the R group compared to the G group. The majority of internal organs were situated within the abdominal plastron in the turtles, where their own weight exerted compressive forces on these organs. In the case of the greenhouse turtles, which exhibited fully aquatic lifestyles, the buoyancy provided by water alleviated this pressure [25]. The rice turtles could proportionally increase the width while decreasing the length of the abdominal plastron, thereby enhancing the contact area between the ventral surface and the ground. This variation alleviated the pressure on internal organs, adapting terrestrial life [26,27]. In turtle fossils, it had been confirmed that the pterygoid bone of the abdomen exhibited a prominent transverse process, which was a characteristic feature of basal reptiles [28]. The BH/BCL of the R group was slightly higher than that of the G and P groups (although not significant), suggesting a relatively more arched shell shape in rice turtles compared to the relatively flatter shell shapes of pond turtles and greenhouse turtles. The relatively flat shell shape could reduce efficiency and hydrodynamic resistance during swimming, facilitating aquatic life. In contrast, the arched shell shape provides robust mechanical support, enabling the organism to withstand significant forces encountered during terrestrial activities, including self-righting and predator defense [27,29,30]. Research has demonstrated that there is a critical threshold for the curvature of a turtle’s shell, which when exceeded results in its stress-bearing capacity being diminished [31,32]. This indicates that the variation in body height between the different groups of Chinese soft-shelled turtles is minimal. Under conditions where stress remains constant, these turtles exhibit optimal shell shape plasticity.

The limbs serve as the primary locomotor organs for Chinese soft-shelled turtles, and their motor behavior adapts in response to changes of habitat. The FLL/BCL and HLL/BCL in the R and P groups were significantly higher than those in the G group, as the pond and paddy field offered more expansive living spaces compared to the greenhouse. There was a positive correlation between limb dimensions and the effective utilization of open space, facilitating the movement of the Chinese soft-shelled turtles in the expansive environment [33,34]. Surprisingly, the FLL/BCL and HLL/BCL were slightly larger in the P group than in the R group (although not significantly). This phenomenon could be attributed to the extensive rice cultivation in the paddy field, which formed a grid-like spatial structure. In response to this environment, Chinese soft-shelled turtles might contract their limbs to minimize interference with rice plant movement, similar to how certain lizards fold their limbs to facilitate movement through grassy or thorny areas [33].

The head and neck are the primary prey-catching organs of turtles, with their adaptability varying according to the food sources available in different cultivating modes. The NL/BCL and BL/BCL of the R group were significantly higher compared to those of the G and P groups, whereas the NB/BCL and HB/BCL were notably lower. This indicates that the head and neck morphology of the greenhouse turtles and pond turtles tended to be relatively shorter, while the head and neck of the rice turtles exhibited a more slender shape. The environment of the greenhouse culturing mode was controlled and secure, where the turtles’ sole food source was commercial feed. In contrast, the paddy field constituted a more expansive ecosystem with a diversity of food resources. On the one hand, the slender neck facilitated a rapid-strike predation strategy, enhancing hunting efficiency. On the other hand, it improved the flexibility of the head movement, allowing for greater steering maneuverability. These morphological adaptations were conducive to exploiting a diverse range of food sources and expansive environment [35,36]. The ID/BCL of the R and P groups was significantly smaller compared to that of the G group, suggesting an adaptive shortening of interocular distance in rice turtles and pond turtles, which enhanced their visual acuity for prey detection. The narrower interocular distance, coupled with longer limbs, aligned with the observed asynchronous evolution of locomotion and vision in vertebrates [37].

4.2. Plasticity of Culturing Modes on Organ Characteristics

The organ indexes of animals serve as a critical indicator of their physiological condition and environmental adaptation. Additionally, the condition factor is a key metric for assessing the growth and development of farmed animals. In this study, LV/BW, VW/BW, IFW/BW, BW/BCL3 and EPW/BCL3 all showed significantly higher measurements in the G group compared to the P and R Groups. Research has demonstrated that the primary source of lipids in turtles is derived from the consumption of commercial feed [38]. The abundant commercial feed provided to the greenhouse turtles, combined with limited activity space due to high-density cultivation, resulted in a lifestyle characterized by overfeeding and restricting movement. This mode accelerated lipid synthesis in the greenhouse turtles, leading to excessive lipid accumulation in the internal organs and limbs, ultimately causing significant obesity. While this condition facilitated weight gain in Chinese soft-shelled turtles, the elevated lipid content might diminish consumer purchase intent due to concerns about quality [39]. Moreover, the high-fat commercial feed induced metabolic impairment, leading to conditions such as fatty liver disease and enteritis [40]. Consequently, the liver and visceral indexes in the G group were significantly higher than those in the other two groups. For the BCW/BW, CW/BW and EPW/BW, the G Group exhibited lower values compared to the P and R Groups, indicating that greenhouse turtles’ body weights primarily increased through lipid accumulation rather than the development of muscle, calipash and back carapace. Moreover, lipid accumulation was not an entirely detrimental adaptation. Research on skeletal muscle activity and obesity in humans has demonstrated that physical disorders and obesity decrease joint flexibility and exacerbate pain [41]. The internal fat deposited in the greenhouse turtles was predominantly accumulated within the limbs, serving to cushion and lubricate the joints. This adaptation enhanced the flexibility of movement and mitigated pain during physical activity [42,43].

While rice turtles and pond turtles were provided with commercial feed in identical proportions, their body weights, internal fat accumulation and condition factors were significantly lower than those of the greenhouse turtles. This could be attributed to the more complex living environment provided by the paddy field and pond for Chinese soft-shelled turtles. Such an environment maintained Chinese soft-shelled turtles’ high sensitivity to environmental changes, potentially leading to stress responses and reductions in food consumption [44]. The alternative food sources available in the paddy field and pond, such as insects, likely decreased the reliance on commercial feed and reduced the accumulation of internal fat in the turtles. In the relatively expansive environment, the principle of “seeking benefits and avoiding harm” guided physical activities, resulting in significant internal fat reduction for both rice turtles and pond turtles [45,46]. The calipashes of Chinese soft-shelled turtles exhibit structural similarities to the lateral fins of squids, which function to assist in locomotion [18]. The CW/BW of the R and P groups was significantly higher than that of the G group, indicating that the calipash differences in Chinese soft-shelled turtles might be more closely associated with movement and living environment rather than the consumption of commercial feed.

The back carapace of the Chinese soft-shelled turtle is primarily composed of bony plates, which serve as an indicator of bone density. The BCP/BCL of the R and P groups were lower than that of the G group, whereas the BCW/BW was higher. This indicates that the bone densities of rice turtles and pond turtles are greater than those of greenhouse turtles. In comparison to the greenhouse, the paddy field and pond were more readily exposed to sunlight. This increased exposure facilitated the synthesis of vitamin D3 through sunbathing, which enhances calcium deposition and ultimately leads to the enhanced bone density of Chinese soft-shell turtles [47,48,49]. Other studies have demonstrated that increased physical activity could enhance bone density [50]. This might also explain the higher bone density observed in rice turtles and pond turtles. The back carapace constitutes the primary skeletal framework of the Chinese soft-shelled turtle, constraining the overall skeletal dimensions and influencing muscle attachment. The BCP/BCL of the G group was significantly higher than that of the R and P groups, whereas the EPW/BW of the G group was notably lower than that of the R and P groups. This suggests that the muscles of rice turtles and pond turtles were relatively heavier with reduced skeletal muscle attachment, indicating a more compact muscle structure [51]. However, the adequate consumption of high-fat commercial feed has been shown to decrease the rate of muscle synthesis, which might explain the relatively relaxed muscles of the greenhouse turtles [40].

4.3. Correlation Between Morphological and Organ Indexes

The correlation analysis of morphological and organ indicators provided a systematic approach to evaluating how changes in morphological plasticity influence behavior and the physiological state of Chinese soft-shelled turtles in different culturing modes. The LV/BW, IFW/BW, BW/BCL³ and EPW/BCL³ were indicators reflecting the physiological state associated with obesity, whereas the ID/BCL reflected the observational power related to food-seeking behavior. In the correlation analysis, organ indicators associated with obesity were positively correlated with physical indicators related to food-seeking observation. The passive consumption of commercial feed resulted in a decline in food-seeking observation, and the morphological characteristics of greenhouse turtles were primarily marked by relatively wide interocular distances. The abundant consumption of commercial feed could lead to the physiological state of obesity. In contrast, the increased food diversity and enhanced food-seeking capabilities in rice turtles and pond turtles led to the formation of relatively narrower interocular distances. Decreasing the consumption of commercial feed and increasing the amount of exercise that promoted “risk avoidance and benefit seeking” led to the relatively robust physiological conditions of rice turtles and pond turtles.

The BCW/BW and EPW/BW could serve as direct indicators of bone density and muscle content in Chinese soft-shelled turtles. In the correlation analysis of organs related to movement, both indexes exhibited a positive correlation with the hind limb and negative correlation with the forelimb (although not significantly). The unique morphological structure of Chinese soft-shelled turtles, the abdominal plastron which resembles the thoracic girdle of lizards, thereby limits the functionality of the fore limbs in locomotion, whereas the hind limbs remain unrestricted [52]. Reptiles adapted to environmental variations by modifying their hind limb movements, increasing both stride length and step frequency, and enhancing the flexibility of their locomotion [53,54]. It has also been confirmed in studies of turtles’ locomotion that the hind limbs provide the vast majority of the propulsive force during terrestrial movement [55]. The rice turtles and pond turtles enhanced their terrestrial adaptability through the development of stronger hind limb mobility, increased flexibility and locomotive capacity, as well as elevated bone density and muscle content. Furthermore, the edible portion index exhibited a positive correlation with the hind limb, suggesting that the hind limb served as the primary site of muscle production.

4.4. Effects of Culturing Modes on Proximate Compositions

Proximate compositions are fundamental criteria for evaluating meat quality. The crude protein content in the muscles of the R and P groups was significantly higher compared to that of the G group, while the crude fat content was notably lower than that of the G group. Rice turtles and pond turtles decreased their consumption of high-fat commercial feed, while increasing locomotion in their expansive spaces. This promoted protein synthesis and prevented fat infiltration into the muscles [56,57,58]. The lifestyle of decreasing their consumption of commercial feed and increasing locomotion would lead them to develop the characteristics of higher protein and lower fat contents in the muscle. The abundant consumption of commercial feed and sedentary lifestyle of greenhouse turtles led to the development of muscles with the characteristics of lower crude protein and higher fat content. The intramuscular fat content is an indicator of muscle juiciness [59]. The higher levels of intramuscular fat of the greenhouse turtles contributed to enhanced meat tenderness. Despite the culturing modes having a certain influence on the proximate compositions of muscle in Chinese soft-shelled turtles, the protein and fat contents of the muscles remained within the ranges of 18.820-20.450 g/100 g and 0.713-0.957 g/100 g, respectively. This indicates that the turtle meat retained the characteristics of being higher in protein and lower in fat, aligning with contemporary dietary preferences [60].

4.5. Effects of Culturing Modes on Amino Acids

The nutritional value of protein is largely determined by the concentration of essential amino acids (EAAs). In this study, the proportion of EAAs/TAAs and EAAs/NEAAs in the muscles of Chinese soft-shelled turtles were found to be 40.453%–41.237% and 67.943%–70.177%, respectively. These compositions fundamentally satisfied the FAO/WHO standard for high-quality proteins in the ideal model, with approximately 40% EAAs/TAAs and over 60% EAAs/NEAAs [61]. This suggests that the muscles of Chinese soft-shelled turtles are a high-quality source of protein. The EAA contents and EAAI indexes of the muscles of the R and P groups were significantly higher than those of the G group, suggesting that the amino acid nutritional value of the rice turtles and pond turtles are superior. Consequently, when consuming an equivalent amount of turtle meat, rice turtles and pond turtles could provide a greater amount of protein.

The savory flavor profile of meat is influenced by the concentration of flavor amino acids (FAAs). Glutamic acid (Glu) and aspartic acid (Asp) are the representative amino acids associated with the umami taste, while glycine (Gly) and alanine (Ala) are key amino acids linked to the sweet taste [62]. The FAA contents of the muscles of the R and P groups were significantly higher than that of the G group, indicating that the meat of rice turtles and pond turtles exhibits superior sweetness and is more delicious.

The healthcare benefits of the Chinese soft-shelled turtle are attributed to the pharmacodynamic amino acids (PAAs) presented in its muscle. This study identified a total of 10 pharmacodynamic amino acids, including Glu, arginine (Arg), Gly, methionine (Met) and Asp, which have been shown to reduce the incidence of cardiovascular diseases, cancer and diabetes [63,64,65,66]. Additionally, lysine (Lys) has been found to prevent recurrence following viral infections [67], while tyrosine (Tyr) and phenylalanine (Phe) serve as precursors for dopamine synthesis, which could alleviate mental stress [68]. The concentrations of PAAs in muscles of the R and P groups were significantly higher than that in the G group, suggesting that the health-promoting effects of rice turtles and pond turtles are superior.

Valine (Val), Isoleucine (Ile) and Leucine (Leu) are three critical branched-chain amino acids (BCAAs) that are indispensable for the synthesis of muscle protein. They play a key role in maintaining skeletal muscle growth and providing energy during locomotion [69,70]. The BCAA contents of the muscles of the R and P groups were significantly higher than that of the G group, suggesting that rice turtles and pond turtles exhibit superior muscle protein synthesis capabilities. This finding is consistent with the observation that the crude protein contents of the muscles of rice turtles and pond turtles exceed that of greenhouse-turtle. Finally, the collagen proportion and collagen content of the calipash are critical factors in evaluating the quality of Chinese soft-shelled turtles. Hydroxyproline, a unique amino acid, is the characteristic amino acid of collagen [71]. The hydroxyproline contents of the calipashes of the R and P groups were significantly higher than that of the G group, indicating a greater collagen content in the calipashes of rice turtles and pond turtles. Additionally, the higher calipash indexes observed in the R and P groups suggested that both rice-turtle co-culturing and pond culturing modes could enhance the edible collagen content.

4.6. Effects of Culturing Modes on Fatty Acids

In the nutritional value evaluation system of fatty acids, the content and composition of UFAs serve as a valuable indicator. In this study, the UFAs/SFAs and PUFAs/SFAs of the turtles’ muscles across three groups were found to be 2.304–2.976 and 0.909–1.355, respectively. These values are consistent with the recommended fatty acid profiles for a healthy diet [72]. Unlike SFAs, most UFAs, particularly PUFAs, are primarily obtained through dietary intake due to the body’s limited or absent capacity for their synthesis. Notably, the muscles of the greenhouse turtles exhibited higher concentrations of UFAs and PUFAs, while demonstrating lower concentrations of SFAs. This could be attributed to the fact that UFAs and PUFAs are essential fatty acids for Chinese soft-shelled turtles, predominantly acquired through their diet [73]. Commercial feed, which contains significant amounts of fish oil, fish meal and vegetable oil, could provide a rich supply of UFAs and PUFAs [74,75,76]. However, the decrease in the consumption of commercial feed led to lower concentrations of UFAs and PUFAs and higher concentrations of SFAs in the muscles of rice turtles and pond turtles.

Based on the position of unsaturated bonds, PUFAs can be further classified into omega-3 (n-3) and omega-6 family PUFAs (n-6) [77]. The key n-3 PUFAs include α-linolenic acid (ALA, C18:3n-3), eicosapentaenoic acid (EPA, C20:5n-3) and docosahexaenoic acid (DHA, C22:6n-3). The key n-6 PUFAs include linoleic acid (LA, C18:2n-6) and arachidonic acid (AA, C20:4n-6). ALA and LA are essential PUFAs for humans, playing crucial roles in reducing fracture risk and maintaining cardiovascular and cerebrovascular health. However, as they cannot be synthesized endogenously, they must be obtained through diet [78]. ALA serves as a precursor for the biosynthesis of other n-3 PUFAs, including EPA and DHA. EPA and DHA have been shown to be beneficial for reducing blood lipids and blood pressure, maintaining cardiovascular and cerebrovascular health and promoting nervous system development [79,80]. LA is a precursor for the biosynthesis of AA. AA is not only crucial for preventing neurological disorders but also plays an essential role in the development of the central nervous system and retina in neonates [81]. In this study, the muscles of the R and P groups exhibited significantly lower concentrations of ALA and LA compared to those of the G group. This observation might be attributed to the fact that turtles, similar to humans, lack the ability to synthesize ALA and LA endogenously and must obtain it through diet. Upon comparison, the ALA content in the muscles of greenhouse turtles was approximately twice that of the other two groups, which appeared to corroborate the hypothesis that these PUFAs originate from commercial feed. The concentrations of EPA, DHA and AA in the muscles of the rice turtles and pond turtles were significantly higher than those of the greenhouse turtles. Besides promoting nervous system and visual development, EPA and DHA also play a crucial role in regulating skeletal muscle protein metabolism [82]. Moreover, studies have demonstrated that AA and DHA are effective at enhancing exercise performance [83]. These findings suggest that the rice turtles and pond turtles, with their adaptive changes in lipid metabolism due to the more complex environments of the paddy field and pond, could maintain superior sensory and motor abilities. Notably, the three PUFA derivatives of EPA, DHA and AA exhibit less variability compared to ALA, indicating that these three PUFAs have a more constrained biosynthetic capacity in Chinese soft-shelled turtles. Although PUFAs confer numerous health benefits, there are antagonistic effects among different types of PUFAs. An imbalanced composition ratio of PUFAs may potentially lead to chronic inflammation. Consequently, the appropriate PUFA ratio serves as a critical indicator for evaluating the nutritional value of fatty acids [84,85]. An n-6/n-3 ratio of PUFAs in the range of 0.55-5 is considered an optimal recommended range [86]. In this study, the n-6/n-3 ratio of PUFAs in the muscles was found to be between 0.610-1.158, all falling within the recommended range. Regarding dietary fatty acid intake, consuming turtle meat could contribute positively to human health.

5. Conclusions

The morphology, organ indexes and nutritional compositions of Chinese soft-shelled turtles exhibited significant variations in response to different culturing modes. The results of this study indicate that Chinese soft-shelled turtles adapt to a terrestrial lifestyle by developing a broader plastron and arched shell shape and adapt to aquatic environments by creating a relatively flattened body shape. The expansive spaces, abundant sunlight and diverse food sources available in the paddy field and pond shaped the morphological characteristics of the rice turtles and pond turtles, resulting in longer limbs, narrower interocular distances and slender necks and heads. Compared to the greenhouse turtles, these turtles also exhibited a robust physiological condition with a higher proportion of carapace, calipash and edible tissues. Ecological culturing modes increased the contents of essential amino acids, flavor amino acids, pharmacodynamic amino acids and collagen, thereby enhancing the nutritional quality of the Chinese soft-shelled turtle. From a fatty-acid perspective, while these ecological culturing modes elevated the levels of EPA+DHA and monounsaturated fatty acids, they also led to a reduction in polyunsaturated fatty acids due to decreased consumption of commercial feed. This partially diminished the nutritional advantages. Therefore, to fully harness the benefits of the ecological culturing modes in enhancing nutritional value, it is recommended that an optimized feeding strategy should be implemented during the ecological cultivation of the Chinese soft-shelled turtle.