A feed for juvenile gudgeon and its application

Abstract

The application discloses a feed suitable for being eaten by juvenile Pungtungia niphes and application thereof, and relates to the technical field of fish feed. The feed comprises the following components: 30-46 parts of soybean meal with skin; 5 parts of rapeseed meal; 5 parts of flour; 18-20 parts of wheat; 1-30 parts of fish meal; 1 part of zeolite powder; 1.5 parts of calcium dihydrogen phosphate; 0.5-1.5 parts of soybean oil; 0.1 part of choline chloride; 1 part of premix; and 0.9 parts of glycine. According to the characteristics of juvenile Pungtungia niphes, the application designs the feed suitable for being eaten by juvenile Pungtungia niphes, and the protein content is about 28-41%. It is found through research that the feed with high protein content can better improve the growth speed of juvenile Pungtungia niphes, and the feed with low protein content can improve the immune biochemical function of juvenile Pungtungia niphes and maintain the intestinal health of juvenile Pungtungia niphes, including maintaining better immune antioxidant function, complete liver and intestinal tissue structure and rich intestinal microbial community, which is beneficial to the long-term healthy growth of juvenile Pungtungia niphes.

Description

Technical Field

[0001] This invention relates to the field of fish feed technology, specifically to a feed suitable for juvenile fine-scaled gudgeon and its application. Background Technology

[0002] Protein is the most essential nutrient in aquatic feed, directly affecting fish growth, immune function, and metabolic health. The appropriate level of protein supplementation varies depending on the fish species and growth stage. Insufficient protein can lead to stunted growth and decreased immunity in fish, while excessive protein can increase the metabolic burden on the liver, causing problems such as lipid deposition and tissue damage, while also resulting in wasted feed costs and nitrogen pollution in the aquatic environment.

[0003] The fine-scaled gudgeon (Plagiognathops microlepis Bleeker), originally named the fine-scaled oblique-jawed gudgeon, belongs to the genus Plagiognathops in the order Cypriniformes and is a small to medium-sized economic fish species found in the Yangtze River basin of my country. This fish is widely used in polyculture, aquaculture, and recreational fishing due to its attractive body shape, tender and delicious flesh, high feed utilization rate, and ability to purify water. Currently, aquaculture production is rapidly increasing, and various feeds are being used, but a dedicated feed for fine-scaled gudgeon is not yet available on the market. Therefore, it is necessary to develop a specialized feed for juvenile fine-scaled gudgeon to optimize their intestinal flora, maintain healthy hepatobiliary tissue, and promote their long-term healthy growth.

[0004] Chinese patent CN104222677A discloses a special feed for juvenile fine-scaled carp and its preparation method. Although the patent provides a formula that closely resembles the fish's natural diet, it uses a large amount of natural aquatic plants such as goldfish algae and water chestnut algae, as well as camphor tree leaves and pine needles. These raw materials not only have seasonality issues, such as the inability to collect fresh raw materials in winter and early spring, making it impossible to carry out feed production continuously throughout the year; but also the crude fiber and crude protein content of plants collected from different places and in different seasons varies greatly, resulting in unstable nutritional composition of each batch of feed, which does not meet the quality control standards of the modern feed industry. Summary of the Invention

[0005] The purpose of this invention is to at least solve one of the technical problems existing in the prior art, and to provide a feed suitable for juvenile fine-scaled gudgeon and its application. Based on the feeding characteristics of fine-scaled gudgeon, this invention designs an isolipid feed with a protein level of approximately 28% to 41%, and provides a nutritionally balanced feed by controlling the crude fat content to approximately 4% and the crude fiber content to approximately 6%, and by providing trace elements through premixes. This feed is suitable for the growth and development of juvenile fine-scaled gudgeon, helps optimize the intestinal flora structure of juvenile fine-scaled gudgeon, maintains the health of the hepatobiliary tissue, and promotes the long-term healthy growth of juvenile fine-scaled gudgeon. Furthermore, the nutritional components of the raw materials in the feed of this invention are not significantly affected by season or place of origin, and the nutritional composition is stable. This invention involves feeding fine-scaled gudgeon separately in the same aquaculture system for 8 weeks, measuring various growth indicators, and collecting serum, intestinal, and muscle tissues to analyze and compare differences in growth performance, muscle nutrition, immune antioxidant capacity, hepatobiliary tissue morphology, and intestinal microbiota. The study found that this feed can effectively improve the growth rate of juvenile fine-scaled gudgeon, and also enhance their immune and biochemical functions and maintain their intestinal health, including maintaining better immune and antioxidant function, intact hepatobiliary tissue structure, and a rich intestinal microbiota, thus promoting the long-term healthy growth of juvenile fine-scaled gudgeon.

[0006] The technical solution of the present invention is as follows: In a first aspect, the present invention provides a feed suitable for juvenile fine-scaled gudgeon, the feed comprising the following components in parts by weight: 30-46 parts of soybean meal with husk; 5 parts of rapeseed meal; 5 parts of wheat flour; 18-20 parts of wheat; 1-30 parts of fish meal; 1 part of zeolite powder; 1.5 parts of calcium dihydrogen phosphate; 0.5-1.5 parts of soybean oil; 0.05-0.15 parts of choline chloride; 0.5-1.5 parts of premix; and 0.8-1 part of glycine. The protein content in the feed is 27wt%~42wt%.

[0007] Optionally, the weight proportions of each component in the feed are as follows: 46 parts soybean meal with husk; 5 parts rapeseed meal; 5 parts wheat flour; 20 parts wheat; 1 part fish meal; 1 part zeolite powder; 1.5 parts calcium dihydrogen phosphate; 1.5 parts soybean oil; 0.1 parts choline chloride; 1 part premix; and 0.9 parts glycine.

[0008] Optionally, the weight proportions of each component in the feed are as follows: 30 parts soybean meal with husk; 5 parts rapeseed meal; 5 parts wheat flour; 18 parts wheat; 30 parts fish meal; 1 part zeolite powder; 1.5 parts calcium dihydrogen phosphate; 0.5 parts soybean oil; 0.1 parts choline chloride; 1 part premix; and 0.9 parts glycine.

[0009] Optionally, the fishmeal has a protein content of 65%; the premix comprises the following components in parts by weight: thiamine 7.5-8.5 parts, riboflavin 8-9 parts, pyridoxine 5-7 parts, cyanocobalamin 0.01-0.02 parts, folic acid 1.2-1.4 parts, calcium carbonate 16-18 parts, inositol 38-42 parts, biotin 0.14-0.16 parts, ascorbic acid 100-120 parts, vitamin A 0.8-1 part, vitamin D 0.03-0.05 parts, vitamin E 35-45 parts, vitamin K 4-5 parts, FeSO4·7H2O 40-50 parts, CuSO4·5H2O 8-12 parts, ZnSO4·7H2O 50-56 parts, KI 1-1.4 parts, MnSO4·4H2O 8-9 parts, CoSO4·4H2O 0.5-1.5 parts.

[0010] Secondly, the present invention provides the application of the feed in at least one of the following: increasing the growth rate of juvenile fine-scaled gudgeon, improving the immune and biochemical function of juvenile fine-scaled gudgeon, and improving the intestinal health of juvenile fine-scaled gudgeon.

[0011] Thirdly, the present invention provides the application of low-protein feed in improving the immune and antioxidant function of juvenile fine-scaled gudgeon, maintaining the intestinal flora structure of juvenile fine-scaled gudgeon, and maintaining the health of the hepatobiliary tissue of juvenile fine-scaled gudgeon.

[0012] Optionally, the application includes at least one of the following: 1) Increase the serum immunoglobulin T content in juvenile fine-scaled gudgeon; 2) Increase the immunoglobulin D content in juvenile fine-scaled gudgeon; 3) Increase the total intestinal protease content of juvenile fine-scaled gudgeon; 4) Increase the α-amylase content in juvenile fine-scaled gudgeon; 5) Reduce malondialdehyde content in the liver of juvenile fine-scaled gudgeon; 6) Reduces the intestinal lipase activity of juvenile fine-scaled gudgeon; 7) Increase the height of intestinal villi and the thickness of muscle layer in juvenile fine-scaled gudgeon.

[0013] Optionally, the application includes at least one of the following: 1) Enhance the diversity of gut microbiota in juvenile fine-scaled gudgeon, including OTU count, ACE index, and Chao1 index; 2) Increase the relative abundance of Verrucomicrobia and Bdellovibrio in the intestines of juvenile Gudgeon at the phylum level; 3) Increase the relative abundance of *Flavobacterium* and *Bdellovibrio* in the intestines of juvenile *Flavobacterium spp.* at the genus level.

[0014] Fourthly, the present invention provides the application of low-protein feed in reducing the deformity rate of juvenile fine-scaled gudgeon.

[0015] Fifthly, the present invention provides the application of high-protein feed in improving the growth rate of juvenile fine-scaled gudgeon, the application including increasing the final body weight, final average body length, weight gain rate and specific growth rate of juvenile fine-scaled gudgeon.

[0016] This invention has at least one of the following beneficial effects: This invention designs a feed specifically for juvenile fine-scaled gudgeon based on their characteristics. By adjusting the amount of fishmeal added, the protein content of the feed is made to be approximately 28%~41%. The crude fat content is controlled to be approximately 4% and the crude fiber content to be approximately 6% by adjusting the amounts of soybean meal, rapeseed meal, flour, wheat, and soybean oil. Suitable trace elements are provided through premixes, thus providing a nutritionally balanced feed with protein, fat, fiber, and trace elements, suitable for the growth and development of juvenile fine-scaled gudgeon. Studies have found that this feed can significantly improve the growth rate of juvenile fine-scaled gudgeon, enhance their immune and biochemical functions, and maintain their intestinal health, including better immune and antioxidant functions, intact hepatobiliary tissue structure, and a rich intestinal microbiota. This is beneficial for the long-term healthy growth of juvenile fine-scaled gudgeon. This invention provides a scientific basis for the research and development of specialized compound feeds for fine-scaled gudgeon and for large-scale healthy aquaculture. Attached Figure Description

[0017] Figure 1 The intestinal tissue morphology of juvenile fine-scaled gudgeon from different feed groups is shown; where PH: fold height, PW: fold width, MT: muscle layer thickness, and CD: crypt depth.

[0018] Figure 2 Intestinal tissue morphology indicators of juvenile fine-scaled gudgeon from different feed groups.

[0019] Figure 3 The liver tissue morphology of juvenile fine-scaled gudgeon from different feed groups is shown in the annotation; where V: vacuoles, S: hepatic sinusoids.

[0020] Figure 4 This is a Venn diagram; where different circle colors represent different groups, overlapping areas represent the number of OTUs common to the groups, and non-overlapping areas represent the number of OTUs unique to each group.

[0021] Figure 5 The relative abundance of intestinal microorganisms in juvenile fine-scaled gudgeon from different feed groups.

[0022] Figure 6 This is a bar chart for the analysis of variance between groups. Detailed Implementation

[0023] To make the technical problems solved, the technical solutions, and the beneficial effects of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0024] Example 1 This embodiment provides a low-protein feed (LP group, crude protein 28%), the composition and weight of which are as follows: 46g soybean meal with husk; 5g rapeseed meal; 5g wheat flour; 20g wheat; 1g fish meal (protein content 65%); 1g zeolite powder; 1.5g calcium dihydrogen phosphate; 1.5g soybean oil; 0.1g choline chloride; 1g premix; 0.9g glycine.

[0025] The premix comprises the following components by weight: thiamine 8mg, riboflavin 8.5mg, pyridoxine 6mg, cyanocobalamin 0.015mg, folic acid 1.3mg, calcium carbonate 17mg, inositol 40mg, biotin 0.15mg, ascorbic acid 110mg, vitamin A 0.9mg, vitamin D 0.04mg, vitamin E 40mg, vitamin K 4.5mg, FeSO4·7H2O 45mg, CuSO4·5H2O 10mg, ZnSO4·7H2O 53mg, KI 1.2mg, MnSO4·4H2O 8.5mg, and CoSO4·4H2O 1.00mg.

[0026] Example 2 This embodiment provides a high-protein feed (HP group, crude protein 41%), the composition and weight percentage of which are as follows: 30g soybean meal with husk; 5g rapeseed meal; 5g flour; 18g wheat; 30g fish meal (protein content 65%); 1g zeolite powder; 1.5g calcium dihydrogen phosphate; 0.5g soybean oil; 0.1g choline chloride; 1g premix; 0.9g glycine.

[0027] The composition and proportion of the premix are the same as in Example 1.

[0028] Example 3 This embodiment provides a feed with a high protein content (LHP group, crude protein 35%), the composition and weight of which are as follows: 30g soybean meal with husk; 5g rapeseed meal; 5g wheat flour; 17.5g wheat; 30g fish meal (protein content 65%); 1g zeolite powder; 1.5g calcium dihydrogen phosphate; 0.5g soybean oil; 0.1g choline chloride; 1g premix; 0.9g glycine.

[0029] The composition and proportion of the premix are the same as in Example 1.

[0030] Comparative Example 1 This comparative example provides a feed with lower protein content (LLP group, crude protein 20%), with the following composition and weight: 22g soybean meal with husk; 5g rapeseed meal; 8g wheat flour; 42g wheat; 3g fish meal (protein content 65%); 1g zeolite powder; 1.5g calcium dihydrogen phosphate; 1.5g soybean oil; 0.1g choline chloride; 1g premix; and 0.9g glycine.

[0031] The composition and proportion of the premix are the same as in Example 1.

[0032] Comparative Example 2 This comparative example provides a high-fat, low-protein feed (HLP group, crude protein 20%, crude fat 10%), with the following composition and weight: 18g soybean meal with husk; 9g rapeseed meal; 38g wheat flour; 33g wheat; 4g fish meal (protein content 65%); 1g zeolite powder; 1.5g calcium dihydrogen phosphate; 8g soybean oil; 0.1g choline chloride; 1g premix; and 0.9g glycine.

[0033] The composition and proportion of the premix are the same as in Example 1.

[0034] Comparative Example 3 This comparative example provides a high-fat, low-protein feed (MHLP group, crude protein 28%, crude fat 10%), with the following composition and weight: 30g soybean meal with husk; 12g rapeseed meal; 18g wheat flour; 10g wheat; 15g fish meal (protein content 65%); 1g zeolite powder; 1.5g calcium dihydrogen phosphate; 5g soybean oil; 0.1g choline chloride; 1g premix; and 0.9g glycine.

[0035] The composition and proportion of the premix are the same as in Example 1.

[0036] Comparative Example 4 This comparative example provides a high-fat, high-protein feed (HHP group, crude protein 35%, crude fat 10%), with the following composition and weight: 42g soybean meal with husk; 16g rapeseed meal; 15g wheat flour; 8g wheat; 12g fish meal (protein content 65%); 1g zeolite powder; 1.5g calcium dihydrogen phosphate; 7g soybean oil; 0.1g choline chloride; 1g premix; and 0.9g glycine.

[0037] The composition and proportion of the premix are the same as in Example 1.

[0038] Example 4 The low-protein feed from Example 1, the high-protein feed from Example 2, the relatively high-protein feed from Example 3, and the feeds from Comparative Examples 1-4 were fed to juvenile gudgeon in the same aquaculture system for 8 weeks. Growth indicators were measured, and serum, intestinal, and muscle tissues were collected to analyze and compare differences in growth performance, muscle nutrition, immune antioxidant capacity, hepatobiliary morphology, and intestinal microbiota. The specific procedures are as follows: 1. Materials and Methods 1.1 Test Materials The study period was from April 15 to June 17, 2024. The fish used in the experiment were purchased from the Guanhui Aquaculture Professional Cooperative in Pingxiang City. They were cultured at the Jiangxi Provincial Fisheries Research Institute using the same aquaculture system (embedded containerized recirculating aquaculture system), with each container measuring 6×2×1.5 m in length. Two types of feed were custom-produced from the company, differing only in protein levels. A summary of the feed's nutritional composition is shown in Table 1. This experiment was designed with an LP group (feed from Example 1) and an HP group (feed from Example 2). Small-scaled gudgeon with an initial body weight of 9.46±2.11 g were randomly divided into two groups, with three replicates per group, and a stocking density of 0.5 kg / m². 3 On March 28, the fine-scaled gudgeon were placed in this breeding system to acclimatize to the environment for 19 days. After the system stabilized, they were fed four times a day at 07:30, 11:30, 15:30 and 19:30, and breeding management was carried out according to the standard.

[0039] Table 1. Feed composition and nutrient levels (air-dried matter) The table below shows the measured values ​​of crude protein, crude fat, and crude fiber for each raw material. 1.2 Methods 1.2.1 Growth performance After 56 days of rearing, samples were taken 24 hours after feeding was stopped. Twenty fish were randomly weighed from each tank. The fish were anesthetized with 100 mg / L MS-222. The body length of each fish was measured using calipers (accuracy 0.01 cm), and the final body weight of each fish was measured using an analytical balance (accuracy 0.01 g). After dissection, the visceral and liver weights were measured. A portion of the liver and kidneys was separated and collected for enzyme activity determination. The calculation formula is as follows: Survival rate of fine-scaled gudgeon (SR, %) = N t / N0×100; Deformity rate = (Number of deformed individuals / Number of surviving individuals) × 100; Weight gain rate (WGR, %) = (W t -W0) / W0×100; Specific growth rate (SGR, % / d) = 100% × [ln(W t )-ln(W0)] / t; Feed conversion ratio (FCR, %) = F / (W t -W0)×100; Protein efficiency (PER, %) = (W t -W 0) / (F×P)×100; Liver body weight ratio (HIS, %) = W g / W t ×100; Visceral ratio (VIS, %) = W v / W t ×100; Fullness (CF, %) = W t / L t 3 ×100.

[0040] In the formula, N t N0 and N0 represent the total number of fish at the end and beginning of the experiment, respectively; W0 represents the fish mass (g) at the beginning of the experiment; W t F represents the fish mass (g) at the end of the experiment; F represents the feed intake mass (g); P represents the crude protein content (%) in the feed; W represents the fish mass (g). g For liver and pancreas mass (g); W v Visceral mass (g); L t The final fish length (cm) represents the total length of the fish in the experiment; t represents the number of days the fish were kept in the water (d). 1.2.2 Determination of muscle nutrient composition Twelve small-scaled gudgeon were selected from each group, and their dorsal muscle tissue was collected and stored at -80℃. According to GB 5009.3-2016, the samples were dried to constant weight in an oven at 105℃, and the moisture content was calculated by the loss-in-weight method. Ash content was determined according to the method specified in GB 5009.4-2016; the samples were placed in a crucible and ignited in a muffle furnace at 550℃ for 4 h. Crude protein content was determined according to GB 5009.5-2016; after acid digestion, the crude protein content was determined using the Kjeldahl method. Crude fat content was determined according to GB 5009.6-2016 using Soxhlet extraction.

[0041] 1.2.3 Serum tissue biochemistry Twelve small-scaled gudgeon were selected from each of the above sampling groups. Blood was collected after anesthesia with MS-222. Blood was drawn from the tail vein using a disposable syringe and placed in centrifuge tubes. After standing at 4°C for 2 hours, the blood was centrifuged at 4000 rpm for 10 minutes at 4°C, and the serum was aliquoted into 1.5 mL centrifuge tubes and stored at -80°C. Blood biochemical indicators were detected using a URIT-8021A fully automated biochemical analyzer (URIT, Guilin). α-amylase (α-AL), total protease, and immunoglobulins T and D were detected using enzyme-linked immunosorbent assay (ELISA). Superoxide dismutase (SOD) and lipase (LPS) were detected using the WST-8 assay. All ELISA kits were purchased from Jianglai Biotechnology Co., Ltd., and other kits were purchased from Nanjing Jiancheng Biotechnology Research Institute. The specific assay methods were strictly performed according to the kit instructions. According to the manufacturer's instructions, the detection kits from Nanjing Jiancheng Biotechnology Institute (Nanjing, China) were used to detect malondialdehyde (MDA) (A003-1) and superoxide dismutase (SOD) (A001-3).

[0042] 1.2.4 Organizational Morphology From each of the above sampling groups, 12 small-scaled gudgeon were selected, and midgut tissue and liver tissue the size of a soybean were harvested. After fixation with paraformaldehyde for 24 hours, dehydration with graded concentrations of ethanol, clearing with xylene, paraffin embedding, sectioning, and hematoxylin-eosin (HE) staining, the tissues were observed and photographed under an upright microscope (AxioScope A1, Zeiss, Germany). The villi length, villiwidth, crypt depth, and muscle thickness of each section were measured and recorded using Image software. Simultaneously, the cellular structure within the liver tissue was observed, and differences in their structural morphology were compared and analyzed.

[0043] 1.2.5 Gut microbial sequencing From each group of sampled fish, 12 fish were placed on an ice tray for dissection. The intestines were cut off with sterile dissecting scissors, and the intestinal contents were collected into sterile tubes. After flash freezing in liquid nitrogen, the tubes were transferred to an ultra-low temperature freezer at -80°C for storage and 16S rDNA assay. Total DNA from intestinal microorganisms was extracted using a fecal micro-DNA extraction kit (OMEGA, USA), and the concentration and purity of the extracted DNA were detected using Nanodrop 2000. Universal primers 338F (5'ACTCCTACGGGAG-GCAGCA3') and 806R (5' GGACTACHVGGGT-WTCTAAT3') were used to amplify the V3-V4 variable regions of the intestinal microorganisms in the samples. The products were then purified, quantified, and homogenized to form sequencing libraries. The constructed libraries underwent quality control. 16S rDNA libraries that passed quality control were sequenced using the Ilumina NovaSeq sequencing platform. Sequencing and analysis were outsourced to Shanghai Baiqu Biomedical Technology Co., Ltd. Trimmomatic v0.33 software was used for quality control of the sequencing results, filtering out adapter sequences, low-quality sequences, and short-read sequences to obtain high-quality sequences. Noise reduction was performed using the dada2 method in QIIME 2, including paired-end sequence splicing and chimeric sequence removal, ultimately yielding non-chimeric sequences. QIIME2 was used to calculate the alpha diversity indices of the samples, including Chao1, Ace, Shannon, Simpson, and Coverage indices. Naive Bayes classifiers were used to perform taxonomic annotation of the feature sequences using the Silva.138 database to obtain the species classification information corresponding to each feature. Community composition was statistically analyzed at the phylum and genus levels for each sample. Species abundance data at the phylum / genus level were generated using QIIME2, and community structure diagrams were plotted using R.

[0044] 1.3 Data Analysis Experimental data were analyzed using IBM SPSS Statistics 25 software, and all data are expressed as mean ± standard error (Mean ± SEM). One-way ANOVA was used to analyze sample differences; for statistically significant differences, Duncan's multiple comparisons were used, and a p-value < 0.05 was considered statistically significant. The experimental results were plotted using GraphPad Prism 5 software.

[0045] 2. Results and Analysis 2.1 Effects of different protein levels in feed on the growth performance of juvenile fine-scaled gudgeon The growth data of juvenile fine-scaled gudgeon after 8 weeks of rearing are shown in Table 2. The final average body weight of the LP group and the HP group were 42.90 and 52.17 g, respectively, and the final average body length was 13.48 and 12.24 cm, respectively. Significance tests showed that the average deformity rate, final average body weight, final average body length, weight gain rate, specific growth rate, visceral ratio, and condition factor of the HP group were significantly higher than those of the LP group (P<0.05). There were no significant differences in visceral ratio and average survival rate between the two groups (P>0.05), while the feed conversion ratio and protein efficiency were significantly lower in the HP group than in the LP group (P<0.05).

[0046] Table 2 Comparison of growth and feed conversion ratio of juvenile fine-scaled gudgeon in different feed groups 2.2 Effects of different protein levels in diet on the physiological and biochemical properties of *Gnaphalium affine* The physiological and biochemical indicators of different groups of fine-scaled gudgeon are shown in Table 3. The significance test showed that the levels of immunoglobulin T and D, total intestinal protease and α-amylase in the LP group were significantly higher than those in the HP group (P<0.05). There were no significant differences between the two groups in liver and kidney superoxide dismutase (SOD) and kidney propylene glycol (MDA) (P>0.05), while liver propylene glycol (MDA) and intestinal lipase were significantly lower in the LP group than in the HP group (P<0.05).

[0047] Table 3 Comparison of serum immune, liver and kidney antioxidant indicators and intestinal digestive enzymes in juvenile fine-scaled gudgeon from different feed groups 2.3 Effects of different protein levels in feed on muscle quality of juvenile fine-scaled gudgeon The muscle composition of juvenile fine-scaled gudgeon is shown in Table 4. The crude fat content of the muscle of the HP group was significantly higher than that of the LP group (P<0.05), while there were no significant differences in moisture, crude protein, and ash content (P>0.05).

[0048] Table 4 Comparison of muscle composition (%, wet weight, n=4) of juvenile fine-scaled gudgeon from different feed groups 2.4 Effects of different protein levels in diet on intestinal morphology of juvenile gudgeon The intestinal tissue morphology and structure of juvenile fine-scaled gudgeon are as follows: Figure 1 As shown. Statistical results of intestinal villus data are as follows. Figure 2 As shown. From Figure 2 It can be seen that the height of the intestinal villus folds and the thickness of the muscle layer in the LP group were significantly higher than those in the HP group (P<0.05), while there were no significant differences in the width of the intestinal villus folds, the depth of the crypts, and the ratio of fold height to crypt depth (P>0.05).

[0049] 2.5 Effects of different protein levels in diet on liver tissue morphology of juvenile gudgeon The morphology and structure of the liver tissue of juvenile fine-scaled gudgeon are as follows: Figure 3 As shown. From Figure 3 It is evident that the hepatic cords of the LP group of fine-scaled gudgeon were clear, and the hepatocytes were intact. In contrast, the hepatocyte nuclei of the HP group of fine-scaled gudgeon were shifted, vacuoles were present, and hepatic sinusoids were increased.

[0050] 2.6 Effects of different protein levels in diet on intestinal microbial diversity in juvenile gudgeon 2.6.1 Gut Microbial Diversity and Venn Diagram Table 5 shows the alpha diversity index of the gut microbiota of juvenile gudgeon. As shown in the table, the ACE and Chao1 indices of the LP group were significantly higher than those of the HP group (P<0.05); there were no significant differences in the Simpson and Shannon indices between the two groups (P>0.05), but the Shannon index value of the LP group was greater than that of the HP group; the Good's coverage index of both groups reached 1, indicating that the sequencing depth of the samples was high and could basically cover all microbial communities in the samples.

[0051] Table 5 Comparison of intestinal microbial diversity in juvenile fine-scaled gudgeon in different feed groups Figure 4 The Venn diagram shows the common and unique microorganisms of the LP and HP groups. As can be seen from the diagram, the LP group contains more OUTs than the HP group, with 6720 and 5847 respectively. The two groups share 2032 OUTs, accounting for 19.29% of all OUTs.

[0052] 2.6.2 Analysis of Gut Microbial Community Composition and Differences Figure 5 The results show that at the phylum level, Proteobacteria was the dominant phylum in all samples. In the LP group, the dominant phyla for LP1 and LP3 also included Planctomycetes and Verrucous Microbes, the dominant phyla for LP2 included Firmicutes and Actinobacteria, the dominant phyla for LP4 and LP5 included Verrucous Microbes and Actinobacteria, and the dominant phyla for LP6 included Planctomycetes and Bacteroidetes. In the HP group, the dominant phyla for HP1 and HP2 included Firmicutes and Bacteroidetes, the dominant phyla for HP3 included Firmicutes and Fusobacterium, the dominant phyla for HP4 included Planctomycetes and Patellar Bacteria, the dominant phyla for HP5 included Fusobacterium and Bacteroidetes, and the dominant phyla for HP6 included Bacteroidetes and Actinobacteria.

[0053] At the genus level, the dominant genera of LP1 include *Pyrophyllus*, *Bacillus*, and *Rhodobacteraceae*; the dominant genera of LP2 include ZOR0006, *Aeromonas*, and *Cetobacter*; the dominant genera of LP3 include *Pyrophyllus*, *unclassified_Xanthomonadaceae*, and *Rhodobacteraceae*; the dominant genera of LP4 and LP5 include *Bacillus*, *Rhodobacteraceae*, and *unclassified_Rhodobacteraceae*; and the dominant genera of LP6 include *Aeromonas*, *Pyrophyllus*, and ZOR0006. The dominant genera of HP1 and HP3 include ZOR0006, Aeromonas, and Cetacea; the dominant genera of HP2 include Aeromonas, ZOR0006, and Auricularia; the dominant genera of HP4 include Piriformis, Bacillus, and Cetacea; the dominant genera of HP5 include Cetacea, Luba Lake Pipe Bacterium, and unclassified_Xanthomonadaceae; and the dominant genera of HP6 include Bacillus, ZOR0006, and Luba Lake Pipe Bacterium.

[0054] Figure 6 A bar chart showing the inter-group variance analysis between the LP and HP groups is presented to test the significance of differences in means among multiple samples. At the phylum level, there were significant differences in the abundance of three phyla between the LP and HP groups: the relative abundance of Verrucous Microbes and Bdellovibrio in the LP group was significantly higher than that in the HP group (P<0.05), and the relative abundance of Fusobacterium in the HP group was significantly higher than that in the LP group (P<0.05). At the genus level, there were significant differences in the abundance of three genera between the LP and HP groups: the relative abundance of Flavobacterium and Bdellovibrio in the LP group was significantly higher than that in the HP group (P<0.05), and the relative abundance of Cetacea in the HP group was significantly higher than that in the LP group (P<0.05).

[0055] 3 Discussion 3.1 Effects of dietary protein levels on the growth performance of juvenile fine-scaled gudgeon Protein is a key limiting nutrient for fish growth, and its intake directly affects protein deposition and growth rate. In this invention, the weight gain rate and specific growth rate of juvenile fine-scaled gudgeon in the HP group (41% crude protein) were significantly higher than those in the LP group (28% crude protein), indicating that high-protein feed can provide sufficient amino acids for the rapid growth of juvenile fish, promoting protein synthesis and weight gain. However, the HP group also showed a significant increase in deformity rate, visceral ratio, and condition factor, suggesting that the excess protein was not fully utilized for growth and was partially converted into fat and deposited in the visceral organs, leading to an increase in visceral index.

[0056] Feed conversion ratio (FCR) and protein efficiency are core indicators for evaluating feed utilization. In this invention, the FCR of the HP group was significantly lower than that of the LP group, but the protein efficiency was significantly reduced. This indicates that *Gymnocypris finniculatus* has a higher energy conversion efficiency for high-protein feeds, but its protein utilization efficiency is lower. This may be because the feed protein level exceeds its suitable requirement (30%-40% for omnivorous fish), and the excess protein is decomposed through deamination to provide energy, resulting in protein waste. Regarding muscle nutrition, only the crude fat content showed differences between groups, indicating that the feed protein level has little impact on the main nutrients such as muscle protein and water in *Gymnocypris finniculatus*. However, high-protein feeds promote fat deposition, which is consistent with the metabolic characteristics of fish where excessive protein is converted into fat.

[0057] 3.2 Effects of dietary protein levels on the immune and antioxidant functions of juvenile fine-scaled gudgeon Fish immune function is closely related to feed nutrition. Appropriate protein levels can enhance the body's immunity, while excessive protein may trigger oxidative stress. Immunoglobulins (IgT and IgD) are the core substances of humoral immunity in fish, and their content directly reflects immune capacity. In this study, the IgT and IgD contents in the LP group were significantly higher than those in the HP group, indicating that low-protein feed is more conducive to maintaining the immune function of juvenile fine-scaled gudgeon, while high-protein feed may inhibit immunoglobulin synthesis.

[0058] Superoxide dismutase (SOD), an important antioxidant enzyme in the body, can scavenge reactive oxygen species (ROS), while microalcoholic acid (MDA) is the end product of lipid peroxidation. Both reflect the level of oxidative stress in the body. In this invention, the liver MDA content in the HP group was significantly increased, while SOD activity did not change significantly. This indicates that the high-protein diet induced oxidative stress in the liver, exceeding the clearance capacity of the antioxidant system, leading to lipid peroxidation damage. As the main organ for protein metabolism, excessive protein metabolic burden on the liver may be a key cause of oxidative stress.

[0059] 3.3 Effects of dietary protein levels on the morphology of hepatointestinal tissue in juvenile fine-scaled gudgeon Intestinal tissue structure and digestive enzyme activity directly affect the efficiency of nutrient digestion and absorption. Intestinal villus height and muscle layer thickness are important indicators for evaluating intestinal absorptive function; higher villus height and thicker muscle layer result in a larger absorption area and stronger digestive function. In this study, the LP group had significantly higher intestinal villus height and muscle layer thickness than the HP group, and significantly higher total protease and α-amylase activities, indicating that a low-protein diet can maintain intestinal structural integrity and enhance digestive enzyme secretion. The decreased digestive enzyme activity in the HP group may be related to intestinal structural damage and intestinal flora imbalance.

[0060] Liver tissue morphology revealed pathological features in the HP group, including vacuolation and nuclear displacement of hepatocytes, indicating that the high-protein diet led to fatty degeneration of the liver. As the core organ for protein metabolism, excessive protein breakdown produces large amounts of harmful substances such as ammonia, increasing the liver's burden and promoting fat synthesis and deposition, ultimately leading to hepatocyte damage. In contrast, the LP group showed intact hepatocyte structure without significant pathological damage, suggesting that a protein level of 28% is more consistent with the liver metabolic capacity of juvenile shad.

[0061] 3.4 Effects of dietary protein levels on the gut microbiota of juvenile fine-scaled gudgeon Gut microbiota participate in fish nutrient metabolism, immune regulation, and gut health maintenance; their diversity and community structure are crucial for host health. Alpha diversity analysis showed that the number of OTUs, ACE index, and Chao1 index in the LP group were significantly higher than those in the HP group, indicating that low-protein diets can maintain higher gut microbiota richness. Higher microbial richness leads to stronger gut functional stability and greater resistance to changes in the external environment and pathogen invasion.

[0062] In terms of community composition, Proteobacteria, Firmicutes, and Bacteroidetes were the core intestinal phyla of the fine-scaled gudgeon, similar to the intestinal flora structure of other freshwater fish such as the yellowtail gudgeon and grass carp, indicating that these phyla are conserved in the intestines of omnivorous fish. Among the phyla showing differences between groups, the relative abundance of Verrucous Microbes and Bdellovibrio was significantly increased in the LP group. Verrucous Microbes are involved in carbohydrate metabolism, and their abundance is closely related to fish health; Bdellovibrio can clear intestinal pathogens and enhance the body's disease resistance. It is speculated that the LP group improved its intestinal health by enriching these two functional phyla. The relative abundance of Fusobacterium was even higher in the HP group. This phylum is involved in protein and fat degradation, which may be adapted to the nutritional characteristics of high-protein feeds.

[0063] At the genus level, *Flavobacterium* and *Bdellovibrio* were enriched in the LP group. *Flavobacterium* has the ability to degrade polysaccharides and can utilize algae and organic detritus, which is suitable for the omnivorous characteristics of *Gymnocypris finnifolia*. *Bdellovibrio*, as a probiotic, can inhibit pathogenic microorganisms and enhance intestinal immunity. The relative abundance of *Cetobacter* was significantly increased in the HP group. This genus can ferment polypeptides and carbohydrates to synthesize vitamin B12, and its enrichment is presumably related to the protein metabolism requirements of high-protein diets. These results indicate that dietary protein levels affect the nutrient utilization and health status of *Gymnocypris finnifolia* by regulating the structure of intestinal functional flora, which is consistent with the general rule that dietary nutrition regulates the intestinal flora of fish.

[0064] 4. Conclusion To determine the optimal dietary protein level for the growth of juvenile *Gnaphalium affine*, this invention designed two isolipid diets: a low-protein group (LP group, crude protein 28%) and a high-protein group (HP group, crude protein 41%). Juvenile *Gnaphalium affine* (initial body weight 9.46 ± 2.11 g) were fed these diets for 8 weeks in a recirculating aquaculture system. Growth performance, muscle nutrient composition, immune and antioxidant indicators, hepatointestinal tissue structure, and intestinal microbiota characteristics were systematically analyzed. Results showed: ① In terms of growth performance, the final body weight, weight gain rate, and specific growth rate of the HP group were significantly higher than those of the LP group (P<0.05), but the deformity rate (5.12% vs 1.01%), visceral ratio (10.53% vs 8.57%), and condition factor (1.81% vs 1.74%) were also significantly higher (P<0.05), and the feed conversion ratio was significantly lower and the protein efficiency was significantly lower than that of the LP group (P<0.05). ② Among the muscle nutrients, only the crude fat content of the HP group (2.44%) was significantly higher than that of the LP group (1.34%) (P), while there were no significant differences in moisture, crude protein, and crude ash (P>0.05). ③ Regarding immune and digestive functions, the serum immunoglobulin T (IgT), immunoglobulin D (IgD), and intestinal total protease and α-amylase activities in the LP group were significantly higher than those in the HP group (P<0.05), while the liver malondialdehyde (MDA) content and intestinal lipase activity were significantly lower in the LP group than in the HP group (P<0.05). There were no differences between the groups in liver and kidney superoxide dismutase (SOD) and kidney MDA content (P>0.05). ④ In terms of tissue morphology, the height of intestinal villi and the thickness of the muscle layer in the LP group were significantly higher than those in the HP group (P<0.05), the hepatic cords were clear and the hepatocyte structure was intact, while the hepatocytes in the HP group showed pathological features such as nuclear shift, vacuolation and increased hepatic sinusoids. ⑤ Regarding gut microbiota, the number of OTUs, ACE index, and Chao1 index in the LP group were significantly higher than those in the HP group (P<0.05), indicating richer microbial diversity. At the phylum level, the relative abundance of Verrucomicrobia and Bdellovibrio in the LP group was significantly higher than that in the HP group, while the proportion of Fusobacteriota was higher in the HP group. At the genus level, Flavobacterium and Bdellovibrio were enriched in the LP group, while the relative abundance of Cetobacterium was significantly increased in the HP group (P<0.05).

[0065] In summary, although the HP group of fine-scaled gudgeon grew faster, it suffered from problems such as a high rate of deformities, liver damage, and weakened immune and digestive functions. In contrast, the LP group, by optimizing the intestinal flora structure and maintaining healthy liver and intestinal tissue, was more conducive to the long-term healthy growth of juvenile fine-scaled gudgeon. This invention provides a theoretical basis for the precise formulation design of artificial feed for fine-scaled gudgeon.

[0066] Therefore, this invention demonstrates that high-protein feed (41%) significantly improves the growth rate of juvenile fine-scaled gudgeon, but leads to increased deformity rate, liver oxidative damage, weakened immune and digestive functions, and reduced intestinal microbial diversity. Low-protein feed (28%), while resulting in slightly slower growth, maintains superior immune and antioxidant functions, intact hepatobiliary tissue structure, and a rich intestinal microbiota, thus promoting the long-term healthy growth of juvenile fine-scaled gudgeon. Considering both growth performance and health status, it is recommended that the crude protein level in the formulated feed for juvenile fine-scaled gudgeon be controlled at around 28%. Further gradient protein levels can be established to precisely select the optimal protein requirement.

[0067] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A feed suitable for juvenile fine-scaled gudgeon, characterized in that, The feed comprises the following components in parts by weight: 30-46 parts soybean meal with husk; 5 parts rapeseed meal; 5 parts wheat flour; 18-20 parts wheat; 1-30 parts fish meal; 1 part zeolite powder; 1.5 parts calcium dihydrogen phosphate; and 0.5-1.5 parts soybean oil. Choline chloride 0.05~0.15 parts; premix 0.5~1.5 parts; glycine 0.8~1 parts; The protein content in the feed is 27wt%~42wt%.

2. The feed according to claim 1, characterized in that, The weight proportions of each component in the feed are as follows: 46 parts soybean meal with husk; 5 parts rapeseed meal; 5 parts wheat flour; 20 parts wheat; 1 part fish meal; 1 part zeolite powder; 1.5 parts calcium dihydrogen phosphate; 1.5 parts soybean oil. Choline chloride 0.1 parts; premix 1 part; glycine 0.9 parts.

3. The feed according to claim 1, characterized in that, The weight proportions of each component in the feed are as follows: 30 parts soybean meal with husk; 5 parts rapeseed meal; 5 parts wheat flour; 18 parts wheat; 30 parts fish meal; 1 part zeolite powder; 1.5 parts calcium dihydrogen phosphate; 0.5 parts soybean oil; Choline chloride 0.1 parts; premix 1 part; glycine 0.9 parts.

4. The feed according to claim 1, characterized in that, The fish meal has a protein content of 65%; The premix comprises the following components in parts by weight: thiamine 7.5-8.5 parts, riboflavin 8-9 parts, pyridoxine 5-7 parts, cyanocobalamin 0.01-0.02 parts, folic acid 1.2-1.4 parts, calcium carbonate 16-18 parts, inositol 38-42 parts, biotin 0.14-0.16 parts, ascorbic acid 100-120 parts, vitamin A 0.8-1 part, vitamin D 0.03-0.05 parts, vitamin E 35-45 parts, vitamin K 4-5 parts, FeSO4·7H2O 40-50 parts, CuSO4·5H2O 8-12 parts, ZnSO4·7H2O 50-56 parts, KI 1-1.4 parts, MnSO4·4H2O 8-9 parts, and CoSO4·4H2O 0.5-1.5 parts.

5. The use of the feed according to any one of claims 1-4 in improving the growth rate of juvenile fine-scaled gudgeon, enhancing the immune and biochemical functions of juvenile fine-scaled gudgeon, and improving the intestinal health of juvenile fine-scaled gudgeon.

6. The use of the feed according to claim 2 in improving the immune and antioxidant function of juvenile fine-scaled gudgeon, maintaining the intestinal flora structure of juvenile fine-scaled gudgeon, and maintaining the health of the hepatointestinal tissue of juvenile fine-scaled gudgeon.

7. The application according to claim 6, characterized in that, The application includes at least one of the following: 1) Increase the serum immunoglobulin T content in juvenile fine-scaled gudgeon; 2) Increase the immunoglobulin D content in juvenile fine-scaled gudgeon; 3) Increase the total intestinal protease content of juvenile fine-scaled gudgeon; 4) Increase the α-amylase content in juvenile fine-scaled gudgeon; 5) Reduce malondialdehyde content in the liver of juvenile fine-scaled gudgeon; 6) Reduces the intestinal lipase activity of juvenile fine-scaled gudgeon; 7) Increase the height of intestinal villi and the thickness of muscle layer in juvenile fine-scaled gudgeon.

8. The application according to claim 6, characterized in that, The application includes at least one of the following: 1) Enhance the diversity of gut microbiota in juvenile fine-scaled gudgeon, including OTU count, ACE index, and Chao1 index; 2) Increase the relative abundance of Verrucomicrobia and Bdellovibrio in the intestines of juvenile Gudgeon at the phylum level; 3) Increase the relative abundance of *Flavobacterium* and *Bdellovibrio* in the intestines of juvenile *Flavobacterium spp.* at the genus level.

9. The application of the feed according to claim 2 in reducing the deformity rate of juvenile fine-scaled gudgeon.

10. The application according to claim 5 or 6, characterized in that, The growth rate includes at least one of body weight, body length, weight gain rate, and a specific growth rate.

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