Method for screening feed protein level of gynocardia acerofolia and application thereof

Abstract

The application provides a method for screening feed protein levels of Xiangyun crucian, comprising the following steps: S1, grouping feeds with different protein contents; S2, continuously feeding the feeds grouped in step S1 to Xiangyun crucians with the same weight respectively; S3, collecting muscle samples from the back of the Xiangyun crucians after the feeding; S4, performing meat quality analysis on the muscle samples, wherein the meat quality analysis comprises carcass and muscle composition analysis, muscle amino acid content analysis, muscle flavor nucleotide content analysis, muscle texture analysis, muscle development-related gene expression level analysis, muscle antioxidant function analysis or / and muscle autophagy state analysis. Compared with the prior art, the screening method of the application is more comprehensive, the obtained data is more accurate, and finally it is confirmed that the feed with a protein level of 29% can improve the muscle quality of Xiangyun crucian.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of aquaculture, and particularly relates to a method for screening a feed protein level of Xiangyun crucian and application thereof. BACKGROUND

[0002] Protein is an important nutrient for aquatic animals, which can affect farmed fish from sources, protein levels, quality and other aspects. The source effect of protein on fish growth performance, meat quality, antioxidant capacity and other aspects is increasingly valued by people. In fact, the content of protein in feed is an effective means to control the cost of aquaculture, and the content of protein in feed also affects the muscle quality of many aquatic animals. For example, the protein content level not only affects the growth performance and meat quality of grass carp, but also changes its antioxidant capacity. The protein level of feed affects the muscle hardness of grass carp by affecting the growth of muscle fibers and the synthesis and degradation of collagen. Low-protein feed will slow down the growth rate of Kuruma shrimp, reduce the activity of intestinal trypsin, and affect the hardness. The protein level and source of feed also affect the antioxidant defense status of rats. High-protein feed may lead to an imbalance between oxidants and antioxidants, thereby inducing oxidative stress in the digestive organs of mice.

[0003] In addition, studies have also found that oxidative stress can have irreversible harmful effects on muscle quality. When animals are stimulated by oxidized fish oil in feed, high-carbohydrate or high-fat feed, and high-temperature environment, the production and clearance of active oxygen free radicals in the body are easily imbalanced, and tissues or cells may thus suffer from oxidative damage. Because polyunsaturated fatty acids in muscle are easily combined with active oxygen free radicals to produce a large amount of lipid peroxidation, especially lipid peroxidation (MDA), the aldehyde group in which can combine with protein residues to activate protein oxidation, resulting in negative effects such as amino acid dysfunction and protease inactivation, thereby impairing muscle nutrition and quality. For example, the inactivation of μ-calpain reduces the amount of myofibrillar protein hydrolysis, resulting in reduced meat tenderness. In addition, excessive accumulation of active oxygen also causes DNA damage in muscle cells, activates poly ADP-ribosyl polymerase 1, and impairs meat tenderness by catalyzing the degradation of muscle fiber connecting proteins. Therefore, reducing the occurrence of oxidative stress in the body and enhancing the defense capacity of the antioxidant system are the key to improving muscle quality.

[0004] Autophagy is the ability of lysosomes to degrade their own components, which is highly conserved. Autophagy levels are highly up-regulated only when the body is under nutritional restriction, copper exposure, pathogenic infection or other stress responses, producing autophagic vesicles for the removal of functionally impaired intracellular membranes, organelles and other substances. Therefore, the level of autophagy can be used as an indicator to reflect the health status of the body. Studies have found that after the body is exposed to oxidative stress, reactive oxygen species (ROS) can activate mitochondrial autophagy to remove damaged mitochondria. However, excessive ROS can damage mitochondrial function, forming a vicious cycle. Studies have shown that there is a possibility of interaction between muscle quality, oxidative stress and the expression of signaling molecules in the autophagy state. Adding antioxidants to the feed can significantly increase the content of polyunsaturated fatty acids in muscle health substances, up-regulate the expression of muscle fiber development-related genes to improve meat hardness, and up-regulate Nrf2-activated antioxidant enzymes while inhibiting LC3 protein expression to alleviate oxidative stress and autophagy.

[0005] Proteins are the most important component in animal feed composition. Due to the difference in digestion and absorption capacity, with the change of feed protein level, the growth performance of animals will be significantly affected through the GH-IGFs axis and the TOR / S6K1 pathway. Due to the enhancement of protein metabolism, animals need to accelerate the formation of ATP to supplement energy, and the content of ROS, the byproduct of ATP production, will also be significantly increased, thereby inducing oxidative damage. Autophagy is closely related to oxidation. Since autophagy is the body's response to oxidative stress, its level will change with the degree of oxidative damage to the body. However, there is little information about the interactive effects of feed protein levels on muscle quality, antioxidant and autophagy.

[0006] Xiangyun crucian carp is a kind of polyploid fish (3n), which has the advantages of fast growth, strong stress resistance and enhanced immune response. Some varieties of Xiangyun crucian carp are produced by crossing male allo-tetraploid with female crucian carp. Compared with the parents, it has some advantages in biological characteristics, such as higher body, shorter tail, smaller head and higher meat yield. In addition, under stress conditions, Xiangyun crucian carp shows stronger stress resistance than diploid crucian carp. In order to further improve the economic value of Xiangyun crucian carp, it is necessary to improve the muscle quality of Xiangyun crucian carp through nutritional strategies. Therefore, it is a technical problem to be solved in the art to seek a suitable feed protein level for Xiangyun crucian carp. SUMMARY

[0007] In view of the deficiencies of the prior art, the purpose of the present application is to provide a method for screening the protein level of Xiangyun crucian carp feed, which analyzes the influence of the protein level of Xiangyun crucian carp feed on its muscle quality at the genetic molecular level, so as to screen out the best protein level.

[0008] In order to achieve the purpose of the present application, the following technical solutions are adopted in the present application:

[0009] In a first aspect, the present application provides a method for screening the protein level of feed for Xiangyun crucian, comprising the following steps:

[0010] S1: grouping the feed with different protein contents;

[0011] S2: continuously feeding the Xiangyun crucian with the same body weight with the feed grouped in step S1 respectively;

[0012] S3: collecting the muscle samples from the two sides of the back of the Xiangyun crucian after the feeding;

[0013] S4: analyzing the meat quality of the samples, which comprises analyzing the carcass and muscle composition, analyzing the amino acid content of the muscle, analyzing the flavor nucleotide content of the muscle, analyzing the muscle texture, analyzing the expression level of muscle development related genes, analyzing the muscle antioxidant function, or / and analyzing the muscle autophagy state.

[0014] Preferably, the different protein contents in step S1 include 26%, 29%, 32%, 35%, 38% or / and 41%.

[0015] Preferably, the step of analyzing the carcass and muscle composition in step S4 is measuring the water content, ash content, and crude fat content in the carcass, or / and measuring the water content, ash content, and crude protein content in the muscle.

[0016] Preferably, the step of analyzing the amino acid content of the muscle in step S4 is measuring the content of glycine, glutamic acid, aspartic acid, tyrosine, alanine, histidine, arginine, threonine, valine, methionine, isoleucine, leucine, lysine, serine, or / and proline in the muscle.

[0017] Preferably, the step of analyzing the flavor nucleotide content of the muscle in step S4 is measuring the content of CMP (cytosine nucleotide), GMP (guanine nucleotide), IMP (inosine nucleotide, also known as inosinic acid), and AMP (adenine nucleotide).

[0018] Preferably, the step of analyzing the muscle texture in step S4 is measuring the muscle texture parameters, which include elasticity parameters, hardness parameters, gumminess parameters, chewiness parameters, adhesiveness parameters, and cohesiveness parameters.

[0019] Preferably, the step of analyzing the expression level of muscle development related genes in step S4 is detecting the expression level of muscle development related genes TOR, IGF1, MyoD, MyHc, MRF4, Myogenin, Myostanin, or / and MEF2A.

[0020] Preferably, the steps of the S4 muscle antioxidant function analysis method are as follows: detecting the expression levels of muscle antioxidant-related genes GSTα, GSTθ, GSTPi, GPX1, GPX4a, Nrf2 and / or Keap1.

[0021] Preferably, the steps of the S4 muscle autophagy state method are: detecting the expression levels of autophagy-related genes PRKAα2, PRKAβ1a, PRKAβ2, PRKAγ1, PRKAγ2b, PRKAγ3, LC3A, LC3β and / or FoxO1A; and the FOXO1 protein level.

[0022] Secondly, the present invention provides a method for regulating muscle development-related genes in crucian carp. The crucian carp are fed with a feed containing 29% protein. After feeding, the expression levels of muscle development-related genes in the muscle tissue of the crucian carp are detected. The muscle development-related genes include TOR, IGF1, MyoD, MyHc, MRF4, Myogenin, Myostanin and / or MEF2A.

[0023] Thirdly, the present invention provides a method for regulating antioxidant-related genes in the muscle of Xiangyun crucian carp. Xiangyun crucian carp are fed with a feed containing 29% protein. After feeding, the expression level of antioxidant-related genes in the muscle of Xiangyun crucian carp is detected. The antioxidant-related genes include GSTα, GSTθ, GSTPi, GPX1, GPX4a, Nrf2 and / or Keap1.

[0024] Fourthly, this invention provides a method for regulating muscle autophagy-related genes in crucian carp. The crucian carp are fed a diet with a protein level of 29%, and the expression levels of muscle autophagy-related genes in the muscle tissue of the crucian carp are detected after feeding. The muscle development-related genes include PRKAα2, PRKAβ1a, PRKAβ2, PRKAγ1, PRKAγ2b, PRKAγ3, LC3A, LC3β and / or FoxO1A.

[0025] Fifthly, the present invention provides a Xiangyun crucian carp feed, wherein the feed has a protein level of 29% and comprises the following components by weight percentage: casein 3.2%, fish meal 12%, soybean meal 20%, rapeseed meal 15%, fish oil 3%, soybean oil 3%, corn starch 21%, wheat flour 10%, choline 0.5%, mineral premix 3%, carboxymethyl cellulose 3%, and cellulose 6.3%.

[0026] Preferably, the mineral premix contains the following components by weight per kg of feed: NaCl, 500.0 mg; MgSO4·7H2O, 8155.6 mg; NaH2PO4·2H2O, 12500.0 mg; KH2PO4, 16000.0 mg; CaHPO4·2H2O, 7650.6 mg; FeSO4·7H2O, 2286.2 mg; C6H 10 CaO6·5H2O, 1750.0 mg; ZnSO4·7H2O, 178.0 mg; MnSO4·H2O, 61.4 mg; CuSO4·5H2O, 15.5 mg; CoSO4·7H2O, 0.91 mg; KI, 1.5 mg; Na2SeO3, 0.60 mg; Corn starch, 899.7 mg.

[0027] In a sixth aspect, the present invention provides a method for improving the muscle quality of Xiangyun crucian carp, the method comprising the following steps:

[0028] (1) Feed Xiangyun crucian carp with the above-mentioned protein level of 29%;

[0029] (2) After the feeding was completed, the Xiangyun crucian carp was dissected and the dorsal muscle was collected and prepared into test samples.

[0030] (3) Detect the expression levels of muscle development-related genes TOR, IGF1, MyoD, MyHc, MRF4, Myogenin, Myostanin and / or MEF2A in the sample of (2);

[0031] (4) Detect the expression levels of muscle antioxidant capacity-related genes GSTα, GSTθ, GSTPi, GPX1, GPX4a, Nrf2 or / and Keap1 in the sample of (2);

[0032] (5) Detect the expression levels of the muscle autophagy state genes PRKAα2, PRKAβ1a, PRKAβ2, PRKAγ1, PRKAγ2b, PRKAγ3, LC3A, LC3β and / or FoxO1A in the sample of (2); and the FOXO1 protein level.

[0033] Preferably, the method for improving the muscle quality of Xiangyun crucian carp further includes determining the moisture, ash, and crude fat content in the carcass; and / or determining the moisture, ash, and crude protein content in the muscle.

[0034] Preferably, the method for improving the muscle quality of Xiangyun crucian carp further includes determining the content of glycine, glutamic acid, aspartic acid, tyrosine, alanine, histidine, arginine, threonine, valine, methionine, isoleucine, leucine, lysine, serine, and / or proline in the muscle.

[0035] Preferably, the method for improving the muscle quality of Xiangyun crucian carp further includes determining the content of CMP, GMP, IMP and AMP.

[0036] Preferably, the method for improving the muscle quality of Xiangyun crucian carp further includes measuring muscle texture parameters, including elasticity parameters, hardness parameters, adhesiveness parameters, chewability parameters, adhesion parameters, and cohesiveness parameters.

[0037] In a seventh aspect, the present invention provides the application of the feed in improving the flavor or quality of Xiangyun crucian carp muscle.

[0038] Eighthly, the present invention provides the application of the feed in improving the antioxidant properties of Xiangyun crucian carp muscle.

[0039] In a ninth aspect, the present invention provides the application of the feed in alleviating the level of autophagy in the muscles of Xiangyun crucian carp.

[0040] In a tenth aspect, the present invention provides the application of Xiangyun crucian carp muscle development-related genes, muscle antioxidant-related genes, and muscle autophagy-related genes in a method for analyzing the protein level in Xiangyun crucian carp feed.

[0041] Preferably, the muscle development-related genes include TOR, IGF1, MyoD, MyHc, MRF4, Myogenin, Myostanin, or / and MEF2A.

[0042] Preferably, the muscle antioxidant-related genes include GSTα, GSTθ, GSTPi, GPX1, GPX4a, Nrf2, or / and Keap1.

[0043] Preferably, the autophagy-related genes include PRKAα2, PRKAβ1a, PRKAβ2, PRKAγ1, PRKAγ2b, PRKAγ3, LC3A, LC3β, or / and FoxO1A.

[0044] Preferably, the method for analyzing the protein level of Xiangyun crucian carp feed further includes detecting the moisture, ash, and crude fat in the carcass of Xiangyun crucian carp; and / or determining the moisture, ash, and crude protein content in the muscle.

[0045] Preferably, the method for analyzing the protein level of Xiangyun crucian carp feed further includes detecting the content of glycine, glutamic acid, aspartic acid, tyrosine, alanine, histidine, arginine, threonine, valine, methionine, isoleucine, leucine, lysine, serine, and / or proline in the muscle of Xiangyun crucian carp.

[0046] Preferably, the method for analyzing the protein level of Xiangyun crucian carp feed further includes detecting the content of CMP, GMP, IMP and AMP in the muscle of Xiangyun crucian carp.

[0047] Preferably, the method for analyzing the protein level of Xiangyun crucian carp feed further includes detecting the texture parameters of Xiangyun crucian carp muscle, including elasticity parameters, hardness parameters, adhesiveness parameters, chewability parameters, adhesion parameters, and cohesiveness parameters.

[0048] Compared with the prior art, the beneficial effects of the present invention are:

[0049] This invention analyzes the impact of protein levels on the muscle quality of Xiangyun crucian carp at the molecular level, from analyzing the nutritional components, amino acid composition, and textural properties of the muscle to the regulatory mechanisms of genes related to muscle development, antioxidant activity, and autophagy. Compared with existing technologies, the screening method of this invention provides a more comprehensive analysis and more accurate data, ultimately confirming that a feed protein level of 29% can improve the muscle quality, antioxidant capacity, and alleviate muscle autophagy in Xiangyun crucian carp. Attached Figure Description

[0050] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used in conjunction with embodiments of the invention to explain the invention and do not constitute a limitation thereof. In the drawings:

[0051] Figure 1 This is a schematic diagram illustrating the changes in flavor nucleotide content in the muscle of Xiangyun crucian carp after 8 weeks of feeding with different protein levels, according to an embodiment of the present invention.

[0052] Figure 2 Bubble graphs of muscle texture of Xiangyun crucian carp after 8 weeks of feeding with different protein levels according to an embodiment of the present invention.

[0053] Figure 3 This is a schematic diagram illustrating the effect of different protein levels in feed on the expression level of muscle development genes in Xiangyun crucian carp according to an embodiment of the present invention.

[0054] Figure 4 This is a schematic diagram illustrating the effect of different protein levels in feed on the gene expression level of the antioxidant system in the muscle of Xiangyun crucian carp according to an embodiment of the present invention.

[0055] Figure 5 This is a schematic diagram illustrating the effect of different protein levels in feed on the expression level of autophagy genes in the muscle of Xiangyun crucian carp, according to an embodiment of the present invention. Detailed Implementation

[0056] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0057] Example 1

[0058] I. Experimental Procedure

[0059] 1. Experimental Design

[0060] Xiangyun crucian carp with an initial body weight of 11.79±0.09g were fed for 8 weeks with diets containing 26%, 29%, 32%, 35%, 38%, and 41% protein, respectively. Each group had 3 replicates, with 30 fish per replicate. After the culture period, the dorsal muscle of 3 Xiangyun crucian carp from each group was randomly collected for crude component determination, texture, flavor substance content determination, muscle quality, antioxidant properties, and autophagy gene and protein expression level detection.

[0061] 2. Pre-experiment acclimatization

[0062] The healthy Xiangyun crucian carp used in this experiment were purchased from the Hunan Provincial Fisheries Research Institute. All fish were placed in the experimental environment for acclimatization for two weeks and fed commercial feed twice a day at 9:00 and 15:00 (32.20% crude protein, 6.54% crude fat, 10.4% ash, and 18.5 MJ / kg total energy). Then, a certain number of healthy Xiangyun crucian carp juveniles of similar size were selected for the experiment.

[0063] 3. Experimental Feed

[0064] Six different isoenergetic feeds with varying crude protein levels were formulated. The feed formulations and chemical compositions are shown in Table 1. The feed ingredients were pulverized and passed through a 40-mesh sieve (0.425 mm in diameter). Subsequently, the ingredients were thoroughly mixed using a mixer with a step-by-step mixing method. The mixture was then extruded into 2 mm diameter pellets using a pellet mill, air-dried, and packaged in sealed bags for storage at -20°C.

[0065] Table 1. Formula and composition of the experimental diet

[0066]

[0067] The above mineral premix formula is as follows (mg / kg feed): NaCl, 500.0 mg; MgSO4·7H2O, 8155.6 mg; NaH2PO4·2H2O, 12500.0 mg; KH2PO4, 16000.0 mg; CaHPO4·2H2O, 7650.6 mg; FeSO4·7H2O, 2286.2 mg; C6H 10CaO6·5H2O, 1750.0 mg; ZnSO4·7H2O, 178.0 mg; MnSO4·H2O, 61.4 mg; CuSO4·5H2O, 15.5 mg; CoSO4·7H2O, 0.91 mg; KI, 1.5 mg; Na2SeO3, 0.60 mg; Corn starch, 899.7 mg.

[0068] 4. Feeding Experiment Design

[0069] Fish were divided into six groups based on different feed protein levels. Fish with an initial body weight of 11.79 ± 0.09 g were housed in 18 recirculating aquaculture system tanks (1.2 mH × 0.8 mD) (30 fish per tank). Each group included three randomly distributed parallel tanks. All fish were acclimatized to the experimental feed for one week. During the rearing experiment, the water temperature was maintained at 24.5 ± 1.0℃, dissolved oxygen content was above 6.5 mg / L, and ammonia nitrogen concentration was <0.5 mg / L. After 8 weeks of rearing, three Xiangyun crucian carp (n = 3 × 3) were dissected from each tank, and dorsal muscle was collected.

[0070] 5. Meat quality analysis

[0071] Three fish were randomly selected from each tank, and the muscle fibers from both sides of the back were collected into 2 ml EP tubes and immediately flash-frozen in liquid nitrogen. The samples were then stored at -80°C for analysis. The samples were dried in an oven at 105°C until constant weight to determine the moisture content. Ash content was determined by ignition in a muffle furnace. Crude protein content was determined by the Kjeldahl method. The content of free amino acids and flavor nucleotides in muscle was determined by high performance liquid chromatography (HPLC) [column: C18 SHISEIDO (4.6 mm × 250 mm × 5 μm); probe: DAD probe; flow rate: 1.0 ml / min; column temperature: 40 ℃; wavelength: 254 nm; mobile phase: A (0.1 mol / L anhydrous sodium: acetonitrile = 97:3, pH 6.5); B (acetonitrile: water = 80:20); gradient elution (Table 2)] [column: Agilent C18 (4.6 mm × 250 mm × 5 μm); probe: DAD probe; flow rate: 1.0 ml / min; column temperature: 25 ℃; wavelength: 254 nm; mobile phase: (phosphate buffer: methanol = 1000:40); isocratic elution].

[0072] Table 2 Mobile phase gradient

[0073]

[0074] Two pieces of dorsal muscle were taken from each fish, cut into uniform thickness and size (1.0cm × 1.0cm × 0.25cm), and their textural properties were analyzed after sampling. Hardness, elasticity, chewiness, adhesion, cohesion, and adhesiveness were measured using TPA mode (TMS-PRO, FTC, America). The initial force ranged from 0.1N to 250N, the probe height was 1cm, and the probe was lowered at a constant speed of 30mm / min, with a deformation of 60% of the original length.

[0075] 6. Real-time quantitative PCR analysis

[0076] Total RNA was extracted using Trizol reagent (Invitrogen, Carlsbad, CA, USA). cDNA was synthesized from 1 μg of total RNA using the Evo M-MLV RT Kit and gDNA Clean for qPCR (Accurate Biology, AG11705), following the manufacturer's instructions. qPCR was performed using the SY BR@Green PreMix Pro Taq HS qPCR Kit (Accurate Biology, AG11701) and a Prism 7500 sequencing system (Applied Biosystems, Foster City, CA, USA) to determine the mRNA expression of different genes. β-actin was used as an internal control. For each sample, three replicates were performed under the following conditions: 95℃ for 30 s, 95℃ for 10 s, and 60℃ for 30 s, for 40 cycles. Relative expression levels were calculated using the 2-ΔΔCt method in Excel. Primers used in this study were designed using Primer Express software, as shown in Table 3.

[0077] Table 3 Primer sequences for real-time quantitative PCR

[0078]

[0079] 7. Immunoblot assay

[0080] Protein expression level analysis was performed as follows: 0.1 g of muscle was added to 1 ml of RIPA lysis buffer and 10 μL of PMSF (Cat. No. SL1020-100 ml, Coolaber, China). The mixture was homogenized at 8000 rpm / min for 10 s, repeated three times. The homogenized solution was placed on ice for 30 min. Then, the solution was centrifuged at 12,000 rpm / min and 4°C for 5 min. The supernatant was transferred to a new 1.5 ml centrifuge tube. Total protein content was determined using a BCA protein assay kit (Cat. No. T9300A, Takara, Japan). Protein was quantified to the same concentration using RIPA lysis buffer and mixed with SDS-PAGE loading buffer (5×) at a 4:1 ratio (Cat. No. P0015L, Beyotime Institute of Biotechnology, China), and denatured at 95°C or higher for 10 min. A total of 40 μg of protein was added to each well and separated by SDS-PAGE using a Mini-ProteanTetra gel electrophoresis system (BioRad, USA). The separated protein was then transferred to a polyvinylidene fluoride membrane (IPVH00010, Millipore Co., USA). After blocking non-specific binding, the membrane was incubated with the primary antibody at room temperature for 120 min, followed by the addition of HRP-conjugated secondary antibody. Finally, protein expression was visualized using the BeyoECL Plus Kit (Cat. No. P0018M, Beyotime Institute of Biotechnology, China). Ponceau S was used for sample volume calibration. Nrf2 antibody (Cat. No. PA5-14144, Invitrogen, USA), Fox01 antibody (Cat. No. PA5-23132, Invitrogen, USA), and GAPDH antibody (Cat. No. SC-47724, Santa Cruz, USA) were used. The immunoblot signal was quantified using Image J.

[0081] 8. Data Statistics and Analysis

[0082] All results are expressed as mean ± SD for different biological samples. The data were first tested for normality and homoscedasticity, then statistically evaluated using one-way ANOVA, and finally Tukey's multiple comparison test was performed using SPSS 18.0. Different lowercase letters indicate statistically significant differences (P < 0.05).

[0083] II. Experimental Results

[0084] 1. Effects of feed protein levels on muscle quality of Xiangyun crucian carp

[0085] 1.1 Effects of dietary protein levels on carcass and muscle composition of Xiangyun crucian carp

[0086] The effects of dietary protein levels on the carcass and muscle composition of Xiangyun crucian carp are shown in Table 4. When the dietary protein level was 35% or higher, the ash content of the fish was higher than in other groups (P<0.05). The muscle moisture content of the group with a dietary protein level of 29% was higher than in other groups. Dietary protein levels had no effect on the carcass moisture and crude fat content, or the muscle ash and crude protein content of Xiangyun crucian carp. Furthermore, different protein levels had no significant effect on condition factor (CF). When the protein level was between 26% and 35%, the hepatosome index showed a decreasing trend, slightly increased at 38%, and finally dropped sharply to its lowest point at 41%, but the differences between groups were not significant (P<0.05).

[0087] Table 4. Carcass, muscle composition (%), and body index of Xiangyun crucian carp after 8 weeks of feeding with different protein levels.

[0088]

[0089] *Flatness (CF) = 100 × Body Width (g) / Body Length (cm) 3

[0090] **Hepatic-to-body ratio (HSI) = 100 × liver weight (g) / body width (g)

[0091] From a macroscopic perspective, the content of nutrients can reflect the quality of fish. Since calcium (Ca) and phosphorus (P) account for approximately 80-90% of the total minerals in fish, ash is mainly composed of inorganic elements such as potassium (K), phosphorus (P), sodium (Na), and calcium (Ca). In this invention, the ash content of Xiangyun crucian carcass increased with increasing feed protein levels, showing a significant positive correlation (r = 0.896, P < 0.05). Increased feed protein levels increased the moisture content of the carcass and muscle while decreasing the fat content. Although the crude fat content of the carcass showed a decreasing trend in this invention, feed protein levels had no significant effect on the moisture and crude fat content of Xiangyun crucian carcass. Furthermore, protein content is a key factor affecting meat quality. Appropriate feed protein levels can significantly increase muscle protein content. In the experiments of this invention, different feed protein levels did not significantly affect the crude protein content of Xiangyun crucian carcass muscle, but when the feed protein level was 29%, the moisture content of Xiangyun crucian carcass muscle significantly increased, indicating that feed protein levels may affect muscle quality by increasing moisture content. On the other hand, a feed protein level of 26-41% has no effect on the crude protein, crude fat, moisture and other nutrients in the carcass and muscle.

[0092] 1.2 Effects of feed protein levels on muscle amino acid and flavor nucleotide content

[0093] Table 5 shows that the content of flavor amino acids fluctuated with increasing feed protein levels, but the overall content showed a trend of first increasing and then decreasing, reaching a peak at the 29% level. The glutamic acid content in fish muscle from high-protein feed was generally higher than that from low-protein feed. There were no significant differences in the contents of alanine, tyrosine, and aspartic acid in the muscle of different groups. Analysis of the effect of feed protein levels on other free amino acids in muscle showed that histidine content was positively regulated by feed protein levels and showed a linear relationship with them. Except for methionine, other free amino acids in Table 5 showed significant differences among different groups, reaching their peak content at appropriate feed protein levels. The content of flavor nucleotides (CMP, GMP, IMP, and AMP) in muscle was less affected by feed protein levels. Figure 1 CMP shows a relatively obvious upward trend.

[0094] Free amino acids in muscle are not only raw materials for muscle protein synthesis but also flavor precursors, significantly influencing the flavor profile of muscle. Six amino acids—glycine, glutamic acid, aspartic acid, tyrosine, phenylalanine, and alanine—are generally considered key to delicious food. Optimal feed protein can improve muscle flavor by altering the content of free amino acids. With increasing feed protein levels, the total amino acid content for muscle flavor increases. The free glutamic acid content in Xiangyun crucian carp muscle showed a fluctuating increasing trend with increasing feed protein levels. The glycine content in Xiangyun crucian carp decreased with increasing protein levels. These results indicate that increasing protein levels generally upregulates the amount of free amino acids in muscle to enhance flavor, but not every amino acid is upregulated. Nucleotides can enhance sweetness and meatiness, and inhibit or eliminate bitterness, sourness, and other undesirable flavors. Nucleotides can also synergize with free amino acids, significantly increasing meat flavor. To date, the effect of feed protein levels on muscle nucleotide content has not been extensively studied, but in this invention, results showed that the CMP content in the 29% protein level group was significantly higher than in other groups, and the IMP content in the 32% protein level group was significantly higher than in other groups. The content of other flavor nucleotides did not differ significantly, which may be a result of rapid nucleotide degradation.

[0095] Table 5. Free amino acid content in the muscle of Xiangyun crucian carp after 8 weeks of feeding with different protein levels.

[0096]

[0097] 1.3 Effects of dietary protein levels on muscle texture

[0098] like Figure 2The textural properties results in Table 6 show significant differences in chewiness and adhesiveness (manifested as different colors). The 32% dietary protein level group exhibited significantly lower muscle chewiness and adhesiveness compared to other groups. Figure 2 ).Note: Figure 2 The results are expressed as mean ± standard deviation (n=6) and analyzed using a t-test. Hardness is expressed as peak load (V15); adhesion is expressed as peak area between two compressions; elasticity is expressed as the difference between two compression lengths (V10-V9); adhesiveness is expressed as hardness * adhesion (V15*V25); cohesion is expressed as the ratio of two compressed areas (V23 / V17); chewiness is expressed as viscous * elasticity (V15*V25*V26).

[0099] Texture characteristics, which mimic the chewing process of food in the mouth, include elasticity, firmness, and chewiness, providing reliable data support for assessing muscle quality. People generally prefer fish with a crisper, firmer, more elastic, and chewier texture. Studies have found that when dietary protein levels increase, muscle cohesion and chewiness initially decrease and then increase. There is a link between nutrient composition and texture. The inventors discovered that while a 29% dietary protein level increased moisture content, it did not significantly weaken muscle firmness; the meat texture may exhibit a delicate and non-loose quality.

[0100] Table 6. Muscle texture parameters of Xiangyun crucian carp after 8 weeks of feeding with different protein levels.

[0101]

[0102] 2. Effects of dietary protein levels on the expression of muscle-related genes in Xiangyun crucian carp

[0103] As attached Figure 3 As shown, dietary protein levels significantly influenced the expression of TOR, IGF1, MyoD, MyHc, Myostanin, and MEF2 mRNA in the muscle of Xiangyun crucian carp. The levels were higher in the 29%–38% dietary protein group than in the 26% and 41% groups, indicating that an appropriate protein level range promoted their expression. Except for Myostanin mRNA, which peaked at 38%, MyoD, MyHc, and Mygenin mRNA levels all peaked at 29%. MRF4 expression was upregulated with increasing dietary protein levels.

[0104] Myofibril development largely determines muscle quality. The MRF family of genes, including MyoD, Myogenin, MRF4, and Myf5, regulates the transcription of key myogenic genes. Myostanin inhibits muscle growth, while IGF1 and MEF2A promote it. Dietary protein levels may activate the TOR signaling pathway, thereby promoting muscle growth. In this invention, when dietary protein levels increased to 29% or higher, the mRNA expression of the protein synthesis-related gene TOR did not increase significantly. Furthermore, dietary protein levels were found to significantly affect muscle development. Excessively high or low dietary protein levels can inhibit the transcription of several important genes for muscle development, stimulate the production of myoglobin-inhibiting hormones, or all three, leading to insufficient muscle development.

[0105] 3. Effects of dietary protein levels on gene expression in the antioxidant signaling pathway of Xiangyun crucian carp

[0106] As attached Figure 4 As shown, the expression of GSTθ and GSTPi mRNA was higher in the 26% dietary protein level group than in other groups, while the 38% and 41% high dietary protein levels promoted the expression of GSTα mRNA in the muscle of Xiangyun crucian carp. The mRNA levels of GPX1 and GPX4a were highest in the 32% dietary protein level group. The expression levels of Nrf2 mRNA and protein decreased with increasing dietary protein levels. Nrf2 and Keap1 showed an antagonistic expression trend in the 38% and 41% protein level groups.

[0107] The body typically strengthens its antioxidant system to combat reactive oxygen species (ROS) generated by protein metabolism, preventing oxidative damage to muscle cells and maintaining overall health. This enhancement primarily occurs by upregulating the mRNA expression of genes such as GSH, GST, and GPX, increasing the activity of enzymes like SOD and GSH, and scavenging oxidative metabolites like ROS and MDA. In this invention, the expression trends of GPx genes (GPx1 and GPx4α) were similar, peaking at 32% dietary protein levels. However, the GST gene exhibited different expression trends with increasing dietary protein levels. The Nrf2-Keap1-ARE signaling pathway is currently the most important endogenous antioxidant signaling pathway in organisms. NRF2 is the most important transcription factor in cellular oxidative stress pathways; through interaction with antioxidant response elements, it can increase the expression levels of various antioxidant proteins and detoxification enzymes, scavenging free radicals generated by oxidative stress and thus maintaining the redox state of cells. In Xiangyun crucian carp, both the mRNA and protein levels of Nrf2 decreased with increasing dietary protein levels, indicating that increased dietary protein levels reduce the antioxidant level of Xiangyun crucian carp. This result was further validated by the expression of keap1, showing that keap1 mRNA expression increased with increasing dietary protein levels. These results reveal a negative correlation between Nrf2 and keap1, indicating that high protein levels may affect the pathway by which Nrf2-Keap1 reduces the antioxidant capacity of Xiangyun crucian carp muscle.

[0108] 4. Effects of dietary protein levels on the expression of genes related to autophagy in Xiangyun crucian carp

[0109] like Figure 5 As shown, in the 32% dietary protein level group, the mRNA expression levels of PRKAα2, PRKAβ1a, PRKAβ2, PRKAγ1, PRKAγ2b, LC3A, LC3β, and FOXO1, as well as the FOXO1 protein expression level, were highest in the muscle of Xiangyun crucian carp. Furthermore, the expression level of the PRKA subtype—PRKAγ3 mRNA—decreased with increasing dietary protein levels.

[0110] Under stress, the body's protective mechanisms are triggered, and a slight increase in autophagy may help cells clear damaged organelles and accelerate metabolism. On the other hand, high levels of autophagy may lead to stress-induced damage, metabolic disorders, and even cell death. In this invention, the mRNA levels of autophagy markers (such as PRKA and LC3 family genes) showed an overall trend of first increasing and then decreasing with increasing dietary protein levels. Since FOXO1 is correlated with autophagy, this invention analyzed the expression trends of FOXO1 genes and proteins and compared them with the mRNA levels of autophagy markers. It was found that autophagy levels first increased and then decreased, reaching a peak at a dietary protein level of 32%. Furthermore, at protein levels of 26-29%, autophagy showed an increasing trend, but the change was not significant, indicating that muscle autophagy was appropriately enhanced, metabolism was normal, and cell function was not impaired. However, at a protein level of 32%, autophagy levels increased significantly. Combined with the previous hypothesis of impaired Nrf2-Keap1 antioxidant function regulation, we determined the possibility of oxidative damage in muscle cells at the 32% protein level group, which means that our previous decline in muscle quality may be related to the increase in autophagy levels. Furthermore, we believe that the downregulation of autophagy-related gene expression in the 38-41% protein level group was a passive rather than an active cellular protective downregulation, indicating that autophagy was suppressed in muscle cells in the high-protein group. 5. Pearson correlation between dietary protein levels and response variables.

[0111] Table 7 shows that the levels of MRF4, histidine, alanine, isoleucine, whole fish ash, LC3β, leucine, and GSTα were highly significantly or significantly positively correlated with feed protein levels. The levels of glycine, adhesiveness, Nrf2 protein, PRKA γ3, HIS, Nrf2, PRKAβ1α, and Fox01 protein were highly significantly or significantly negatively correlated with feed protein levels. Other variables did not show significant relationships with feed protein levels.

[0112] Table 7. Pearson correlation between feed protein levels and response variables.

[0113]

[0114] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. The application of a feed for Xiangyun crucian carp in improving the flavor or quality of Xiangyun crucian carp muscle, characterized in that, The feed for Xiangyun crucian carp increases the content of aspartic acid, threonine, lysine, serine, proline and cytosine nucleotides in the muscle of Xiangyun crucian carp, and decreases the content of glutamic acid, tyrosine, histidine and guanine nucleotides in the muscle of Xiangyun crucian carp. The Xiangyun crucian carp feed increases the moisture content in the muscle. The protein level of the Xiangyun crucian carp feed is 29%, and the feed is composed of the following components by weight percentage: casein 3.2%, fish meal 12%, soybean meal 20%, rapeseed meal 15%, fish oil 3%, soybean oil 3%, corn starch 21%, wheat flour 10%, choline 0.5%, mineral premix 3%, carboxymethyl cellulose 3%, and cellulose 6.3%. The mineral premix consists of the following components by weight per kilogram of feed: NaCl, 500.0 mg; MgSO4·H2O, 8155.6 mg; NaH2PO4·2H2O, 12500.0 mg; KH2PO4, 16000.0 mg; CaHPO4·2H2O, 7650.6mg; FeSO4·7H2O, 2286.2 mg; C6H 10 CaO6·5H2O,1750.0mg; ZnSO4·7H2O, 178.0 mg; MnSO4·H2O, 61.4 mg; CuSO4·5H2O, 15.5mg; CoSO4·7H2O, 0.91 mg; KI, 1.5 mg; Na2SeO3, 0.60 mg; Corn starch, 899.7 mg.

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