Comparison of Muscle Texture Characteristics and Nutritional Composition Between Gene-Edited Intermuscular-Bone-Free Crucian Carp and Other Varieties

Abstract

The intermuscular-bone-free crucian carp (Carassius auratus, WUCI), developed through CRISPR/Cas9-mediated bmp6a and bmp6b knockout, offers advantages in consumer acceptance and processing efficiency. However, its effects on muscle texture and nutritional quality have not been fully elucidated. In this study, we compared the F3 generation of WUCI with its sibling wild-type crucian carp (Carassius auratus, WT), Songpu silver crucian carp (Carassius gibelio var. Songpu, SPYJ), and Fangzheng silver crucian carp (Carassius gibelio var. Fangzheng, FZYJ), focusing on muscle texture characteristics and nutritional attributes. WUCI exhibited significantly higher shear force but lower hardness than most comparison groups, with no substantial differences in muscle fiber morphology. Amino acid profiles were similar among all groups. WUCI showed lower crude fat content than WT but higher than FZYJ, and comparable levels of polyunsaturated fatty acids (PUFA), n-3 fatty acids, docosahexaenoic acid (DHA), and eicosapentaenoic acid (EPA) to WT and SPYJ. These findings indicate that the knockout of bmp6a and bmp6b does not alter muscle mass in crucian carp, supporting the potential of WUCI for commercial adoption as a gene-edited consumer-oriented fish product.

1. Introduction

With the improvement of living standards worldwide, consumers are placing increasing emphasis on the eating quality of aquatic products, particularly the texture, flavor, and nutritional composition of fish meat [1,2]. Fish muscle quality is a comprehensive trait encompassing physical properties (such as color, texture, and microstructure), nutritional components (including crude protein, amino acids, and fatty acids), and sensory attributes (such as flavor and tenderness) [3,4]. Among these, muscle texture is a key criterion for evaluating fish meat quality. It can be quantitatively assessed using Texture Profile Analysis (TPA), which measures parameters such as hardness, elasticity, and chewiness, thereby providing a scientific basis for the quality evaluation of both fresh and processed aquatic products [5,6].

Aquatic products are an important source of high-quality animal protein, essential amino acids (EAA), and n-3 polyunsaturated fatty acid (n-3 PUFA), contributing to a balanced diet and promoting human health [7]. Crucian carp (Carassius auratus) is among the most widely farmed freshwater species in China, valued for its delicious taste, rich nutritional value, and strong environmental adaptability [8]. However, its flesh contains numerous fine and heterogeneous intermuscular bones, which may lodge in the throat, reducing consumer acceptance and hindering processing applications [9]. To address this issue, our laboratory identified bmp6 as a key regulator in intermuscular bone development through multi-omics screening and confirmed in zebrafish that bmp6 knockout eliminates intermuscular bones without compromising muscle or skeletal development [10,11,12,13,14]. Building on these findings, Kuang et al. successfully generated an F3 generation of wholly boneless crucian carp (WUCI) through the CRISPR/Cas9-mediated knockout of bmp6a and bmp6b in diploid crucian carp [15] and demonstrated that there were no significant differences (p > 0.05) in muscle nutritional composition or texture compared with its sibling wild-type controls.

However, as an emerging technology, gene editing food still faces challenges in commercialization and market acceptance. The public is widely concerned about ethical, biosafety, and regulatory issues [16]. Therefore, systematically evaluating the nutritional quality of gene-edited aquatic products, such as intermuscular-bone-free crucian carp, is not only a scientific necessity, but also a critical step in promoting industrial application and alleviating consumer concerns. While Kuang et al.’s study primarily focused on gene function validation and general performance changes, a systematic analysis of the effects of intermuscular bone removal via gene editing on muscle texture and nutritional composition remains lacking. Therefore, in the present study, we systematically compared muscle texture properties and nutritional profiles of four crucian carp strains—WUCI, its sibling wild-type (WT), Fangzheng silver crucian carp (Carassius gibelio var. Fangzheng), and Songpu silver crucian carp (Carassius gibelio var. Songpu)—representing distinct genetic backgrounds and breeding histories. The findings aim to provide a scientific basis for assessing the nutritional quality of gene-edited fish and to inform breeding strategies for improving crucian carp meat quality and developing premium aquatic products.

2. Materials and Methods

2.1. Experimental Fish

The experimental fish included WUCI, WT, SPYJ, and FZYJ, all of which were reared at the Hulan Experimental Station of Heilongjiang River Fisheries Research Institute, Chinese Academy of Fishery Sciences (HRFRI). The culture procedure for these strains was described in our previous work [17] and is summarized here. All four strains were induced to spawn simultaneously by intraperitoneal injection with specific oxytocic agents, including luteinizing-hormone-releasing hormone analogue (LRH-A2, 4 μg/kg body weight; Sansheng, Ningbo, China), domperidone (DOM, 1 mg/kg body weight; Sansheng), and human chorionic gonadotropin (HCG, 100 U/kg body weight; rp192055, Aladdin, Shanghai, China). Fertilized eggs were incubated in hatching cages within a recirculating water system maintained at a constant temperature of 22–23 °C, with dissolved oxygen above 8 mg/L. After hatching, fry were reared at equal densities and fed Artemia nauplii four times daily. When the fry reached a body length of 2–3 cm, 600 individuals of each strain were randomly selected and transferred to a 500 m² outdoor pond for further rearing. Dissolved oxygen was maintained above 6 mg/L, and fish were fed the same commercial pellet diet twice daily.

2.2. Texture Measurement

Muscle texture was characterized using seven parameters: hardness, adhesiveness, elasticity (springiness), cohesiveness, chewiness, gumminess, and shear force. Hardness denotes the peak compressive force required to deform the muscle tissue; adhesiveness represents the work needed to overcome the attractive forces between the muscle surface and the contacting probe; elasticity (springiness) describes the extent to which the muscle returns to its original shape after deformation; cohesiveness reflects the internal bonding and the ability of the muscle to maintain structural integrity during chewing; chewiness quantifies the energy required to masticate the muscle to a swallowable state (typically calculated as hardness × cohesiveness × elasticity); gumminess indicates the force needed to break down the muscle to a swallowable consistency (hardness × cohesiveness); and shear force serves as a key indicator of tenderness, capturing the muscle’s susceptibility to fracture and its perceived juiciness during mastication [18,19]. After 4 months of culture, 30 fish from each strain were randomly selected for the experiment trail. Following anesthesia with 100 mg/L MS-222 (E107465, Aladdin), body length and weight were recorded, and dorsal muscle samples were collected for texture analysis. For texture measurement, dorsal muscle tissue located at the anterior end of the dorsal fin was dissected. After removing the skin, muscle blocks of approximately 4 cm × 1.5 cm × 1.5 cm were prepared. From these, subsamples measuring 1 cm × 1 cm × 1 cm were used to determine hardness, cohesiveness, springiness, gumminess, and chewiness, while subsamples measuring 2 cm× 1 cm× 1 cm were used to determine shear force. Texture properties were measured using a TMS-Pro texture analyzer (Food Technology Corporation, Sterling, VA, USA). The specific testing parameters for hardness, cohesiveness, springiness, gumminess, and chewiness were as follows: A TMS-75mm cylindrical polymethyl methacrylate probe (75 mm diameter) was used, with a pre-test speed of 2.0 mm/s, test speed of 1.0 mm/s, post-test speed of 2.0 mm/s, compression ratio of 30%, trigger force of 0.10 N, and an interval of 5.0 s between measurements. For shear force testing, a TMS lightweight blade set probe was employed, with a maximum force sensing range of 250 N, return distance of 35 mm, and test speed of 30 mm/min. Test conditions were set according to Du et al.’s study [20], with 30 fish of each species measured. The mean values were calculated and reported as final results.

2.3. Muscle Fiber Histological Analysis

Fish samples used for histological analysis were subsampled from those collected for texture measurement. Five fish from each strain were randomly selected for muscle fiber histological examination following the method described by He et al. [21]. Dorsal muscle tissue was dissected using the same procedure described above. Muscle specimens measuring approximately 5 mm × 5 mm × 5 mm were fixed in paraffin solution and transversely sectioned at a thickness of 6 μm. The sections were stained with hematoxylin-eosin (HE, G1120, Solarbio, Beijing, China) for structural observation of muscle fibers using a biological microscope (BX53, OLYMPUS, Tokyo, Japan). The muscle fiber area and density were quantified using Fiji image processing software (400× magnification) [22]. Muscle fiber diameter was determined as the minimum Feret diameter (MinFeret ≥ 10 μm) measured at 0.5 degree rotation intervals. Muscle fiber density was calculated as the total number of intact muscle fibers within the measurement range divided by the total area of intact muscle fibers within the same range. For each specimen, five microscopic fields were imaged and used to calculate the fiber diameter and density; the mean value from the five microscopic fields was taken to represent the values for the corresponding strain.

2.4. Muscle Nutritional Composition Determination

After texture measurements, the remaining muscle tissues were used to determine nutritional composition. For each strain, muscle tissues from 10 fish were pooled to obtain a 300 g composite sample, and 3 pooled samples were prepared and analyzed. The contents of moisture, crude ash, crude protein, crude fat, amino acids, and fatty acids were determined according to the corresponding Chinese national standards, as follows [23]: moisture content was measured by the drying method (GB 5009.3-2016), crude ash by incineration (GB 5009.4-2016), crude protein by the Kjeldahl method (GB 5009.5-2016), crude fat by Soxhlet extraction (GB 5009.6-2016), amino acid composition by an LA8080 automatic amino acid analyzer (Hitachi High-Tech Corporation, Japan) (GB 5009.124-2016), and fatty acid composition by a 7890A gas chromatograph (Agilent Technologies, USA) (GB 5009.168-2016). The nutritional composition analysis was carried out by Qingdao Sci-tech Innovation Quality Testing Co., Ltd.

2.5. Muscle Nutrition Evaluation

The amino acid score (AAS), chemical score (CS), and essential amino acid index (EAAI) of muscle protein were calculated according to the scoring standards proposed by FAO/WHO and based on the whole-egg protein model [24]. The formulas are as follows:

AAS = (amino acid content of sample to be tested, mg/g N)/(FAO/WHO standard content of the same amino acid, mg/g N);

CS = (amino acid content of the sample to be tested, mg/g N)/(content of the same amino acid in whole-egg protein, mg/g N);

$$\mathrm{EAAI}=\sqrt[n]{\left(100A/AE\times 100B/BE\times ···\times 100G/GE\right)}\times 100;$$

where n is the number of EAA compared; A, B, C, …, G represent the EAA contents in crucian carp muscle protein; and AE, BE, CE, …, GE represent the EAA contents in whole-egg protein.

2.6. Statistical Analysis

Results for muscle texture parameters and nutritional components across the four crucian carp strains are reported as mean ± standard deviation (SD). Data were tested for normality prior to analysis using the Shapiro–Wilk test. Statistical analyses were conducted using SPSS 20.0. Group comparisons were performed with one-way analysis of variance (ANOVA), and when significant differences were detected, Tukey’s honestly significant difference (HSD) post hoc test was applied. Spearman’s rank correlation coefficient (n = 30) was used to assess associations among texture characteristics, given the widespread use of principal component analysis (PCA) in meat science to evaluate texture profiles and identify key determinants of meat quality [25,26]. PCA was employed to extract the principal factors underlying texture variation. A two-sided p value < 0.05 was considered statistically significant.

3. Results

3.1. Comparison of Muscle Fiber Structure

Thirty fish per strain were randomly selected for the trial. The mean body weight and total length for each strain were as follows: WUCI, 87.70 ± 19.74 g and 134.69 ± 9.67 mm; WT, 70.60 ± 12.43 g and 124.29 ± 6.82 mm; SPYJ, 82.09 ± 18.19 g and 132.31 ± 8.52 mm; and FZYJ, 75.51 ± 17.67 g and 128.54 ± 10.50 mm (Figure 1A,B). The transverse muscle fiber structures of the four crucian carp strains are shown in Figure 1C. The mean muscle fiber area, from largest to smallest, is as follows: SPYJ 2577.76 ± 193.15 μm², FZYJ 2384.01 ± 57.08 μm², WT 2153.40 ± 171.84 μm², and WUCI 2088.44 ± 533.46 μm². The mean muscle fiber density, from largest to smallest, was WUCI 506.59 ± 136.60 fibers/mm², WT 466.73 ± 36.77 fibers/mm², FZYJ 419.65 ± 9.99 fibers/mm², and SPYJ 389.66 ± 28.82 fibers/mm². Statistical analysis indicated that there were no significant differences in muscle fiber density or area among the four groups (Figure 1D–F).

3.2. Comparison of Muscle Texture Characteristics

The analysis of muscle texture parameters revealed that shear force, hardness, adhesiveness, cohesiveness, springiness, gumminess, and chewiness in the four crucian carp strains ranged from 5.98 to 6.77, from 24.72 N to 29.28 N, from 5.79 to 9.61, from 0.32 to 0.36, from 0.58 to 1.01 mm, from 8.83 to 9.23 N, and from 4.77 to 8.72, respectively. The shear force of WUCI was significantly higher than that of WT, FZYJ, and SPYJ. The hardness of WUCI was significantly lower than that of WT (p < 0.05) but significantly higher than that of FZYJ and SPYJ. The adhesiveness of WUCI was significantly higher than that of FZYJ and SPYJ, with no significant difference compared to WT. No significant differences were found among the four groups in terms of cohesiveness, springiness, gumminess, and chewiness (Figure 2).

Spearman’s rank correlation analysis of muscle texture parameters among the four strains showed strong positive correlations among hardness, adhesiveness, springiness, and chewiness (0.80 ≤ ρ ≤ 1.00), with the highest positive correlation observed between adhesiveness and springiness (ρ = 0.99) (Figure 3). Cohesiveness and gumminess exhibited a strong positive correlation (0.50 ≤ ρ < 0.80). Cohesiveness showed strong negative correlations with adhesiveness and springiness (−0.80 < ρ ≤ −0.50) and weak negative correlations with shear force, hardness, and chewiness (−0.50 < ρ ≤ −0.30). Shear force was weakly and positively correlated with hardness, adhesiveness, springiness, and chewiness (0.30 ≤ ρ < 0.50). No significant correlations were found among the remaining texture parameters (0 ≤|ρ| < 0.30).

The PCA of muscle texture parameters from the four strains identified three principal components, PC1 49.67%, PC2 21.85%, and PC3 14.46%, with a cumulative variance contribution rate of 85.98% (Figure 4A). PC1 was positively correlated with springiness, chewiness, adhesiveness, and hardness and negatively correlated with cohesiveness. PC2 was positively correlated with gumminess and cohesiveness, whereas the other five texture characteristics showed contributions to PC2 below the average loading value (Figure 4B). The overall distribution of the four strains in the PC1-PC2 space was concentrated, indicating limited differences among strains. However, FZYJ and SPYJ exhibited relatively broader distribution ranges, reflecting greater intra-group variance (Figure 4C). The muscle texture profiles of WUCI and WT were the most similar and were encompassed within the distribution ranges of FZYJ and SPYJ.

3.3. Conventional Nutritional Composition of Muscle

The moisture, ash, crude protein, crude fat, and total saccharide contents in the muscle of the four crucian carp strains range from 71.80% to 76.13%, from 1.07% to 1.20%, from 18.43% to 18.80%, from 1.40% to 2.57%, and from 0.87% to 1.50%, respectively (Figure 5). WUCI exhibited moisture content second only to FZYJ; crude protein second only to WT; lower crude fat than SPYJ and WT; and the lowest ash and total saccharide contents among the four crucian carp strains.

3.4. Muscle Amino Acid Composition and Evaluation

The composition and content of amino acids in the muscles of the four crucian carp strains are presented in

Table 1. Muscle amino acid content of four crucian carp varieties. n = 3; $\overline{x}$ ± SD; %.

Amino Acid (g/100 g)	FZYJ	SPYJ	WT	WUCI
Thr *	0.66 ± 0.05 a	0.64 ± 0.02 ab	0.59 ± 0.01 c	0.62 ± 0.02 ab
Val *	0.73 ± 0.05	0.73 ± 0.02	0.68 ± 0.02	0.70 ± 0.03
Met *	0.30 ± 0.02	0.27 ± 0.08	0.30 ± 0.06	0.29 ± 0.06
Leu *	1.20 ± 0.09 a	1.16 ± 0.03 ab	1.08 ± 0.03 b	1.12 ± 0.04 ab
Ile *	0.64 ± 0.05	0.63 ± 0.02	0.61 ± 0.01	0.61 ± 0.02
Lys *	1.46 ± 0.09 a	1.37 ± 0.03 ab	1.30 ± 0.03 c	1.41 ± 0.06 a
Phe *	0.67 ± 0.05	0.65 ± 0.02	0.62 ± 0.02	0.63 ± 0.01
His	0.51 ± 0.04	0.49 ± 0.02	0.47 ± 0.02	0.48 ± 0.01
Arg	0.82 ± 0.06	0.80 ± 0.06	0.74 ± 0.02	0.77 ± 0.04
Gly #	0.78 ± 0.04	0.82 ± 0.12	0.71 ± 0.05	0.72 ± 0.03
Ala #	0.91 ± 0.06 a	0.87 ± 0.05 ab	0.80 ± 0.02 b	0.84 ± 0.03 ab
Pro	0.47 ± 0.03	0.49 ± 0.07	0.43 ± 0.01	0.45 ± 0.01
Tyr	0.45 ± 0.04	0.42 ± 0.02	0.41 ± 0.02	0.43 ± 0.02
Ser	0.58 ± 0.05 a	0.53 ± 0.03 ab	0.50 ± 0.02 b	0.52 ± 0.01 ab
Asp #	1.52 ± 0.11 a	1.45 ± 0.03 ab	1.36 ± 0.03 b	1.41 ± 0.04 ab
Glu #	2.04 ± 0.17 a	1.91 ± 0.09 ab	1.78 ± 0.06 b	1.87 ± 0.08 ab
Cys	0.10 ± 0.01	0.10 ± 0.01	0.10 ± 0.01	0.11 ± 0.03
EAA	6.19 ± 0.40 a	5.94 ± 0.13 ab	5.65 ± 0.09 b	5.86 ± 0.22 ab
NEAA	8.17 ± 0.58 a	7.87 ± 0.47 ab	7.31 ± 0.20 b	7.60 ± 0.27 ab
TAA	13.85 ± 0.95 a	13.32 ± 0.58 ab	12.49 ± 0.27 b	12.98 ± 0.48 ab
UAA	5.25 ± 0.37 a	5.05 ± 0.29 ab	4.65 ± 0.16 b	4.84 ± 0.17 ab
EAA/TAA (%)	41.01 ± 0.11	40.94 ± 0.98	41.48 ± 0.35	41.45 ± 0.36
EAA/NEAA (%)	69.52 ± 0.32	69.34 ± 2.77	70.89 ± 1.01	70.80 ± 1.06

Note: The data were presented as mean ± SD (n = 3). Values with different letters represent significant difference at p < 0.05. *, EAA; #, UAA.

. A total of 17 amino acids were detected in all four strains, including 7 EAA and 10 non-essential amino acids (NEAA). The FZYJ strain exhibited the highest total amino acid content (TAA), EAA content, NEAA content, and umami amino acid content (UAA), all of which were significantly higher than those in the WT strain (p < 0.05). The ratio of EAA to TAA (EAA/TAA) in muscles ranged from 40.94% to 41.48%, with no significant differences among strains (p > 0.05); the ratio of EAA to NEAA (EAA/NEAA) ranged from 69.34% to 70.89%, also without significant differences (p > 0.05).

Lysine had the highest AAS (1.27–1.45) and CS (0.98–1.12) among the EAAs in all strains, whereas methionine plus cystine had the lowest AAS (0.57–0.62) and CS (0.32–0.35) (

Table 2. Evaluation of the nutritional value of EAA in the muscle of four crucian carp varieties.

Essential Amino Acid	FAO/WHO Standard	Egg Protein	FZYJ			SPYJ
			Content (mg/g N)	AAS	CS	Content (mg/g N)	AAS	CS
Ile	250	331	217.34	0.87	0.66	214.74	0.86	0.65
Leu	440	534	406.53	0.92	0.76	393.31	0.89	0.74
Thr	250	292	224.1	0.90	0.77	215.87	0.86	0.74
Val	310	441	247.75	0.8	0.56	246.38	0.79	0.56
Met + Cys	220	386	135.81	0.62	0.35	125.11	0.57	0.32
Phe + Tyr	380	565	379.5	1.00	0.67	363.92	0.96	0.64
Lys	340	441	494.37	1.45	1.12	464.51	1.37	1.05
EAAI			66.43			63.88
Essential Amino Acid	FAO/WHO Standard	Egg Protein	WT			WUCI
			Content (mg/g N)	AAS	CS	Content (mg/g N)	AAS	CS
Ile	250	331	202.79	0.81	0.61	204.24	0.82	0.62
Leu	440	534	359.04	0.82	0.67	375.00	0.85	0.7
Thr	250	292	197.25	0.79	0.68	207.59	0.83	0.71
Val	310	441	227.17	0.73	0.51	235.49	0.76	0.53
Met + Cys	220	386	130.54	0.59	0.33	133.37	0.61	0.35
Phe + Tyr	380	565	342.42	0.9	0.61	354.91	0.93	0.63
Lys	340	441	432.18	1.27	0.98	473.21	1.39	1.07
EAAI			60.24			62.75

). These values indicated that methionine plus cystine was the first limiting amino acid, and valine was the second limiting amino acid in all strains. The EAAI values ranged from 60.24 to 66.43, with FZYJ having the highest EAAI value (66.43) and WT the lowest (60.24).

3.5. Analysis of Muscle Fatty Acid Composition and Content

A total of 17 fatty acids were identified in the muscle tissues of the four crucian carp strains comprising four saturated fatty acids (SFAs), five monounsaturated fatty acids (MUFAs), and eight polyunsaturated fatty acids (PUFAs) (

Table 3. Muscle fatty acid composition and contents of four crucian carp strains.

Fatty Acid (g/100 g)	FZYJ	SPYJ	WT	WUCI
SFA	0.15 ± 0.02 c	0.35 ± 0.00 b	0.42 ± 0.03 a	0.43 ± 0.05 a
C14:0	0.00 ± 0.00 b	0.01 ± 0.00 a	0.01 ± 0.00 a	0.01 ± 0.00 a
C15:0	0.004 ± 0.000 b	-	0.005 ± 0.000 a	0.004 ± 0.000 b
C16:0	0.11 ± 0.01 c	0.24 ± 0.00 b	0.31 ± 0.03 a	0.32 ± 0.03 a
C18:0	0.04 ± 0.00 b	0.09 ± 0.00 a	0.09 ± 0.00 a	0.09 ± 0.01 a
MUFA	0.21 ± 0.03 b	0.59 ± 0.03 a	0.68 ± 0.10 a	0.71 ± 0.06 a
C16:1	0.02 ± 0.00 b	0.05 ± 0.00 a	0.06 ± 0.01 a	0.06 ± 0.00 a
C18:1n9c	0.17 ± 0.02 b	0.50 ± 0.03 a	0.58 ± 0.09 a	0.61 ± 0.05 a
C20:1	0.01 ± 0.00 b	0.04 ± 0.00 a	0.03 ± 0.00 a	0.04 ± 0.00 a
C22:1n9	0.005 ± 0.001	0.004 ± 0.000	0.006 ± 0.002	0.005 ± 0.001
C24:1	0.003 ± 0.002	-	0.001 ± 0.002	-
PUFA	0.14 ± 0.01 c	0.30 ± 0.02 b	0.43 ± 0.10 a	0.37 ± 0.03 ab
C18:2n6c	0.08 ± 0.00 d	0.15 ± 0.00 c	0.28 ± 0.06 a	0.21 ± 0.02 b
C18:3n3	0.01 ± 0.00 b	0.03 ± 0.00 a	0.03 ± 0.01 a	0.03 ± 0.00 a
C18:3n6	-	-	0.002 ± 0.004	-
C20:2	0.00 ± 0.00 b	0.01 ± 0.00 a	0.01 ± 0.00 a	0.01 ± 0.00 a
C20:3n6	0.00 ± 0.00 c	0.01 ± 0.00 b	0.02 ± 0.00 a	0.02 ± 0.00 a
C20:4n6	0.01 ± 0.00 b	0.03 ± 0.00 a	0.03 ± 0.01 a	0.03 ± 0.00 a
C20:5n3 (EPA)	0.00 ± 0.00 b	0.02 ± 0.00 a	0.02 ± 0.00 a	0.01 ± 0.00 a
C22:6n3 (DHA)	0.02 ± 0.00 b	0.04 ± 0.01 a	0.05 ± 0.02 a	0.05 ± 0.00 a
n3	0.04 ± 0.00 b	0.09 ± 0.01 a	0.09 ± 0.03 a	0.09 ± 0.01 a
n6	0.10 ± 0.01 c	0.20 ± 0.01 b	0.33 ± 0.07 a	0.27 ± 0.02 ab
n3/n6	0.35	0.44	0.28	0.35
TFA	0.50 ± 0.06 c	1.24 ± 0.04 b	1.53 ± 0.23 a	1.51 ± 0.13 a
SFA/TFA(%)	30.28 ± 1.29	28.09 ± 1.02	27.61 ± 2.56	28.68 ± 1.28
MUFA/TFA(%)	41.36 ± 1.17 c	47.89 ± 1.31 a	44.65 ± 0.45 b	46.98 ± 0.13 a
PUFA/TFA(%)	28.36 ± 1.59 a	24.02 ± 1.43 b	27.74 ± 2.91 ab	24.34 ± 1.41 b

Note: The data were presented as mean ± SD (n = 3). Values with different letters differ significantly at p < 0.05. n3 represents the total sum of n-3 fatty acids; n6 represents the total sum of n-6 fatty acids.

). SFA content in WUCI and WT was significantly higher than in SPYJ and FZYJ (p < 0.05). MUFA content in WUCI, WT, and SPYJ was significantly greater than in FZYJ (p < 0.05). WT and WUCI also exhibited significantly higher PUFA levels than FZYJ (p < 0.05). Total fatty acid (TFA) content was significantly elevated in WT and WUCI compared with SPYJ and FZYJ (p < 0.05).

4. Discussion

4.1. Analysis of Muscle Texture Characteristics

Muscle texture is a key quality attribute for evaluating the quality and acceptability of fresh and processed foods. It is particularly important for muscle products [27,28]. Numerous studies on aquatic animals have shown that higher muscle fiber density and smaller fiber diameter lead to greater hardness and chewiness and improved tenderness, which, in turn, enhance mouthfeel, muscle quality, and human digestibility and absorption. Conversely, lower density and larger diameter are linked to poorer qualities [3,29,30]. In this study, WUCI had the highest muscle fiber density, whereas SPYJ had the lowest. However, the differences in muscle fiber density among the four strains were not statistically significant (p > 0.05), indicating that the muscle texture at the muscle fiber structural level was similar across strains and that bmp6 knockout did not interfere with muscle fiber development. In addition, muscle fiber area was inversely related to density, which is consistent with typical characteristics of fish muscle fibers under normal conditions [31].

Texture parameters, including hardness, springiness, chewiness, adhesiveness, cohesiveness, gumminess, and shear force, are key indicators of muscle texture [32,33]. In this study, WUCI exhibited a significantly higher shear force than WT, indicating tougher flesh. No significant differences were detected between these two strains for the remaining texture parameters (p > 0.05), suggesting a broadly similar overall texture profile. Compared to WT, FZYJ and SPYJ showed higher cohesiveness but lower hardness, adhesiveness, springiness, and chewiness, implying better structural integrity but a less favorable chewing mouthfeel. Muscle textural traits, including shear force, hardness, and water content, are closely related to the microstructure of muscle fibers, particularly inter-fiber spacing. Wang et al. reported that the higher water holding capacity of common carp (Cyprinus carpio) muscle is primarily attributed to greater myofiber density, which forms a more compact intercellular network that more effectively traps and retains water [34,35]. Increased myofiber density is typically accompanied by reduced fiber diameter, thereby increasing muscle hardness [36]. Overall, texture analysis indicated that WUCI and WT had a softer and more tender texture that SPYJ and FZYJ, although WUCI showed a higher shear force than WT.

PCA revealed largely overlapping clusters among the four strains, indicating limited variation in texture characteristics across strains. WT and WUCI clustered most closely, likely because WUCI is a gene-edited derivative of the WT parental population and therefore shares a similar genetic background. This pattern suggests that the knockout of bmp6a and bmp6b did not markedly affect muscle texture in WUCI. Relative to FZYJ, SPYJ exhibited tighter within-group dispersion and greater sample consistency, consistent with its gynogenetic origin from FZYJ (maternal parent), which tends to yield more uniform trait expression. The differences between FZYJ and SPYJ likely reflect selective breeding processes used to develop SPYJ. In particular, the more heterogeneous genetic background of FZYJ may contribute to greater variability in textural characteristics [37,38].

4.2. Analysis of Conventional Nutritional Components

Moisture, protein, and lipid contents in the proximate composition of fish muscle are key indicators of nutritional value. Fish species with lower moisture content typically exhibit relatively higher protein and lipid levels [39,40]. In this study, the moisture content of WUCI was significantly higher than that of the SPYJ and WT (p < 0.05). Consistent with the inverse moisture–lipid relationship, the crude lipid content of WUCI was significantly lower than that of WT (p < 0.05). The crude protein content of WUCI was also lower than that of WT, although the difference was not significant (p > 0.05). The crude protein content of the four strains ranged from 18.43% to 18.80%, exceeding values reported for several commercially important species, including yellow river common carp (Cyprinus carpio haematopterus) [41], blunt snout bream (Megalobrama amblycephala) [42], and spinyhead croaker (Collichthys lucidus) [43]. The crude fat content of the four strains ranged from 1.40% to 2.57%, all higher than those reported for bighead carp (Hypophthalmichthys nobilis) [44] and yellow river common carp [45]. According to Ackman’s classification [46], FZYJ can be considered a lean fish, whereas SPYJ, WT, and WUCI fall within the low-fat category. Overall, these strains combine relatively high protein with low-to-moderate lipid levels, highlighting their values as daily protein sources.

4.3. Analysis of Amino Acid Composition, Content, and Nutritional Value

Amino acid composition is a key metric for evaluating fish muscle protein quality [47]. Amino acids contribute to sour, bitter, umami, sweet, and salty sensations [48]. Histidine (His), typically perceived as bitter and slightly sour, was lower in the four strains than that in grass carp (Ctenopharyngodon idella) [49], the cold-resistant red purse common carp strain (Cyprinus carpio var. ‘Red purse cold-resistant’), Songpu common carp (Cyprinus carpio var. Songpu), and other fish species [50]. Among the major taste-active amino acids, glycine (Gly) and alanine (Ala) elicit sweetness, while aspartic acid (Asp) and glutamic acid (Glu) confer umami in the presence of sodium salts. The ratio of taste-activate amino acids to total amino acids (UAA/TAA) in the four strains was comparable to that of common carp, bighead carp, spotted sea bass (Lateolabrax maculatus), and amur bream (Parabramis pekinensis) [51], indicating a desirable flavor profile. According to the FAO/WHO ideal protein model, EAA should exceed 40% of TAA, and EAA/NEAA should exceed 60% [52]. In this study, the four strains met these criteria (EAA/TAA > 40%; EAA/NEAA > 60%), supporting their classification as high-quality dietary protein sources with substantial nutritional value.

Based on AAS and CS scores, lysine had the highest score among EAAs in muscle across all four strains. As the first limiting amino acid in cereals, lysine is crucial for balanced nutrition, supporting nervous system function, and promoting growth and development [53,54]; thus, these crucian carp strains are good dietary sources for meeting lysine requirements. In all four strains, the first limiting amino acid was methionine + cysteine and the second was valine, which is consistent with findings in triangular carp (Cyprinus multitaeniata) [55], Chinese hooksnout carp (Opsariichthys bidens) [56], and other species; accordingly, formulated diets should be supplemented with these limiting amino acids. The EAAI, which is an indicator of protein quality that is positively correlated with protein utilization efficiency [57,58], was 62.75 for WUCI, lower than FZYJ (66.43) and SPYJ (63.88) but higher than WT (60.24), indicating a moderate level of protein quality in WUCIs.

4.4. Analysis of Fatty Acid Composition and Content

Fatty acids are a major constituent of fish muscle after water and proteins, serving primarily as metabolic energy sources [38]. Fatty acid composition and levels are key indicators of lipid quality and nutritional value. In the four crucian carp strains, SFA, MUFA, and PUFA accounted for 27.61–30.28%, 41.36–47.89%, and 24.02–28.36% of TFA, respectively. These values fall within the typical range reported for Chinese freshwater fish [59,60], indicating profiles characteristic of freshwater species. Among SFAs, palmitic acid (C16:0) was most abundant, which is consistent with observations in grass carp, common carp, blunt snout bream, northern snakehead (Channa argus), and other freshwater fish [61].

PUFAs have well-documented roles in cardiovascular health and in modulating inflammation and autoimmunity [62,63]. Across the four strains, PUFA content was highest in WT, followed by WUCI, SPYJ, and FZYJ; WT and WUCI did not differ significantly. This pattern suggests that bmp6 knockout has minimal impact on muscle fatty acid composition, preserving lipid nutritional value comparable to WT. The n-3 and n-6 PUFAs are essential fatty acids that mammals cannot synthesize de novo [64]. FAO/WHO recommend a dietary n-3/n-6 ratio (calculated as the total n-3 fatty acid content divided by the total n-6 fatty acid content) of at least 0.1–0.2; ratios above this threshold are associated with reduced blood lipids and lower cardiovascular risk [65]. The n3/n6 ratios of the four strains ranged from 0.28 to 0.44, exceeding the FAO/WHO recommendation and indicating favorable fatty acid profiles with potential health benefits. Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) contribute to anti-inflammation activity, improved cardiovascular function, and support for muscle and neural tissue repair [66,67,68]. EPA and DHA contents in WUCI, WT, and SPYJ did not differ significantly (p > 0.05) but were significantly higher than in FZYJ (p < 0.05), suggesting that consuming muscle from WUCI, WT, and SPYJ may confer greater health benefits than FZYJ.

4.5. Effects of bmp6 Knockout on Muscle Development and Metabolism

The comprehensive profiling of muscle texture and nutrient composition indicated that WUCI was broadly comparable to WT; however, amino acid and fatty acid metabolism were altered in WUCI. For example, levels of threonine (Thr), lysine (Lys), and C18:2n6c were significantly higher in WUCI than in WT, accompanied by slight changes in muscle fiber density and cross-sectional areas. These findings are consistent with the metabolomic analysis reported by Kuang et al. [15], who observed that the metabolites in WUCI muscle were significantly enriched in pathways related to amino acids (e.g., tyrosine, glycine, threonine), fatty acid (e.g., linoleic acid, arachidonic acid) metabolism, steroid hormone biosynthesis, purine metabolism, the citrate (tricarboxylic acid, TCA) cycle, and fatty acid degradation.

As a member of the transforming growth factor-β (TGF-β) superfamily, bmp6 plays a critical role in muscle development and protein and lipid metabolism [69]. Bmp6 has been shown to promote myogenic differentiation and myoblast fusion by activating the SMAD signaling pathway [70] and upregulating muscle-specific transcription factors (e.g., MyoD and Myf5), thereby enhancing muscle protein synthesis [71]. This pathway also suppresses the expression of protein degradation genes, contributing to muscle growth and homeostasis [72]. With respect to lipid metabolism, bmp6 has been reported to promote adipogenesis: bmp6 stimulation induces adipogenic commitment and differentiation [73]. BMPs, as new insulin sensitizers, enhance glucose uptake in mature 3T3-L1 adipocytes via PPARγ and GLUT4 upregulation. Thus, bmp6 knockout in WUCI is likely to perturb processes governing muscle development and metabolism.

Despite these molecular alterations, WUCI exhibited muscle texture and nutrient profiles similar to WT, which may be reflect functional compensation by other BMP family members. Genetic compensation following loss of gene function is common [74]. For example, using CRISPR/Cas9 to knockout bag3 in zebrafish, Diofano et al. found that skeletal muscle architecture and cardiac development remained intact in homozygous mutants; subsequent analyses revealed a significant upregulation of an ortholog, bag2, potentially providing a compensatory function [75]. By analogy, the loss of bmp6 function in fish and other aquatic animals may be compensated by other BMPs (e.g., bmp2, bmp4, bmp7) or by key regulators in downstream signaling pathways. Future studies examining the consequences of bmp6 loss or altered expression on muscle phenotype should integrate multi-omics approaches, including transcriptomics, proteomics, and metabolomics, to delineate the bmp6 regulatory network and compensation mechanisms and thereby clarify its complex contribution to muscle quality.

This study demonstrates that the WUCI strain has muscle quality and nutritional attributes comparable to or surpassing those of other crucian carp strains, highlighting the potential of CRISPR/Cas9-mediated bmp6 knockout to eliminate intermuscular bones in aquaculture. Nevertheless, the widespread deployment of a gene-edited strain may constrict the genetic base of cultured populations, thereby diminishing resilience to emerging diseases, environmental perturbations, and climate variability. Future research and commercialization should adopt rigorous genetic management to preserve adequate genetic diversity while maximizing economic returns. Consumer acceptance and regulatory challenges also remain major barriers to the commercialization of gene-edited aquatic products [76]. Public awareness and national regulatory frameworks continue to evolve; international bodies such as the European Food Safety Authority (EFSA) and FAO/WHO are actively assessing the safety of gene-edited products and developing guidance [77]. Future work should elucidate a bmp6-regulated metabolic pathway, ensure compliance with relevant domestic and international regulations, and prioritize breeding innovative aquaculture varieties that satisfy market demands while achieving broad acceptance across ecological, ethical, and regulatory domains.

5. Conclusions

In this study, we conducted a comprehensive assessment of muscle texture and nutritional quality in WUCI, WT, FZYJ, and SPYJ. The four crucian carp strains exhibited broadly similar muscle fiber architecture and textures; WT and WUCI are more delicate and tender, whereas FZYJ and SPYJ had chewier muscles. The protein composition and content of WUCI did not differ significantly from those of the other three strains, all of which had high-quality sources for dietary protein. The fatty acid composition and content of WUCI were similar to those of WT and SPYJ and significantly more favorable than those of FZYJ, which is consistent with advantages in texture and nutrient profile. This study demonstrates that the CRISPR/Cas9-mediated knockout of bmp6 in Carassius auratus successfully yields fish lacking intermuscular bones with no detectable adverse effects on muscle quality. These findings provide a scientific basis for evaluating the nutritional quality of gene-edited fish. Although no adverse nutritional or textural effects were observed, further studies are needed to assess the long-term and ecological impacts of gene editing.