Composite functional feed for repairing the damage of carassius auratus carbonate alkali

Abstract

The present application relates to the technical field of feeding of crucian carp, and discloses a compound functional feed for repairing carbonate alkali damage of crucian carp, which is composed of alpha-ketoglutaric acid, linoleic acid, laurylene and basic feed, wherein the weight ratio of the alpha-ketoglutaric acid accounts for 1.5% of the total weight of the compound functional feed for repairing carbonate alkali damage of crucian carp, and the weight ratio of the linoleic acid accounts for 0.7% of the total weight of the compound functional feed for repairing carbonate alkali damage of crucian carp. The compound functional feed for repairing carbonate alkali damage of crucian carp can significantly enhance the repair capacity of the tissue cells of the crucian carp, improve the disease resistance of the crucian carp, reduce the risk of diseases and promote the energy metabolism process of the crucian carp, so as to ensure the efficient use of energy and maintain the normal homeostasis of the body.

Description

Technical Field

[0001] This invention relates to the field of crucian carp feeding technology, specifically a compound functional feed for repairing carbonate-alkali damage in crucian carp. Background Technology

[0002] In recent years, frequent droughts and floods caused by global climate change, intense evaporation, and seawater intrusion have led to severe land salinization worldwide, resulting in an expansion of saline-alkali water areas that continue to increase. This further compresses freshwater aquaculture space, and carbonate-type waters within saline-alkali waters are rich in high levels of HCO3-. - It is one of the common stressors faced by aquatic organisms. It can alter the pH of water, thereby increasing the concentration of NH3 in the water. This will seriously affect the growth, metabolism, and development of aquatic organisms, ultimately leading to a reduction in biomass and significant changes in species composition.

[0003] To improve the utilization efficiency of carbonate-type saline-alkali water resources and combine aquaculture with saline-alkali waters to form a water-salt-organism ternary symbiotic model, crucian carp need to be raised in carbonate-type saline-alkali waters. However, crucian carp are a major economic freshwater fish, and their cultivation in carbonate-type saline-alkali waters can lead to problems such as energy metabolism imbalance and immune function suppression. Therefore, in order to improve the survival rate of crucian carp in carbonate-type saline-alkali waters, it is necessary to propose a compound functional feed for repairing carbonate-alkali damage in crucian carp. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a compound functional feed for repairing carbonate-alkali damage in crucian carp, thus solving the problems mentioned in the background section.

[0005] The present invention provides the following technical solution: a compound functional feed for repairing carbonate-alkali damage in crucian carp, wherein the compound functional feed for repairing carbonate-alkali damage in crucian carp comprises α-ketoglutaric acid, linoleic acid, myrcene and a basic feed.

[0006] The α-ketoglutaric acid accounts for 1.5% of the weight of the compound functional feed used to repair carbonate-alkali damage in crucian carp.

[0007] The linoleic acid accounts for 0.7% of the weight of the compound functional feed used to repair carbonate-alkali damage in crucian carp.

[0008] The myrcene accounts for 0.5% of the weight of the compound functional feed used to repair carbonate-alkali damage in crucian carp.

[0009] Preferably, the basic feed includes fish meal, soybean meal, wheat, and rice bran.

[0010] Preferably, the nutritional composition of the basic feed is as follows: crude protein ≥29.0%, crude fat ≤12.0%, crude ash ≤15.0%, crude fiber ≥5.0%, moisture ≤12.5%, lysine ≥1.4%, and total phosphorus ≥0.6%.

[0011] Preferably, the basic feed is a solid particle that can be pulverized and passed through a 60-mesh sieve.

[0012] Preferably, the compound functional feed for repairing carbonate-alkali damage in crucian carp is a solid pellet feed with a pellet diameter of 2 mm.

[0013] Preferably, the compound functional feed for repairing carbonate-alkali damage in crucian carp is used to feed crucian carp raised in saline-alkali waters.

[0014] Preferably, the compound functional feed for repairing carbonate-alkali damage in crucian carp is used to protect the integrity of the liver tissue structure of crucian carp under salt-alkali stress, so that the liver plates are arranged regularly, the liver cells are uniform, the morphology is normal, and the cell boundaries are clear.

[0015] Preferably, the compound functional feed for repairing carbonate-alkali damage in crucian carp promotes energy balance, cell growth and development, immune regulation, and inhibition of inflammatory factors.

[0016] Preferably, the compound functional feed for repairing carbonate-alkali damage in crucian carp is used to reduce the difference in gene expression levels of the AMPK signaling pathway in the liver of crucian carp, and to inhibit protein degradation and excessive apoptosis of hepatocytes.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] 1. This compound functional feed for repairing carbonate and alkali damage in crucian carp significantly enhances the repair capacity of crucian carp tissue cells, improves disease resistance, reduces the risk of disease occurrence, and promotes the energy metabolism process of crucian carp by adding functional components such as α-ketoglutarate, linoleic acid, and myrcene to the nutrients, thereby ensuring efficient energy utilization and maintaining normal homeostasis of the body.

[0019] 2. This compound functional feed for repairing carbonate-alkali damage in crucian carp improves the survival efficiency of crucian carp in carbonate-type saline-alkali waters, thereby forming a three-element symbiotic model of water-salt-organisms in saline-alkali waters. This combines aquaculture with saline-alkali waters, improves the efficiency of saline-alkali water resource utilization, and increases the yield of crucian carp in this environment. Attached Figure Description

[0020] Figure 1 A schematic diagram of the histological changes in the liver of a crucian carp;

[0021] Figure 2 Cluster heatmap of differential metabolites in the livers of crucian carp in groups C and CY;

[0022] Figure 3 Cluster heatmap of differential metabolites in the livers of crucian carp in groups T and TY;

[0023] Figure 4 Clustering heatmap of differential metabolites in the livers of crucian carp in groups F and FY;

[0024] Figure 5 A diagram illustrating the metabolic pathway analysis of differentially metabolized substances in crucian carp liver;

[0025] Figure 6 A schematic diagram showing the GO annotation and classification of differentially expressed genes in crucian carp.

[0026] Figure 7 This is a schematic diagram of GO enrichment analysis of crucian carp DEGs. Detailed Implementation

[0027] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0028] Example 1:

[0029] Please see Figure 1 A compound functional feed for repairing carbonate-alkali damage in crucian carp, the compound functional feed for repairing carbonate-alkali damage in crucian carp comprises α-ketoglutaric acid, linoleic acid, myrcene and a base feed.

[0030] The α-ketoglutaric acid accounts for 1.5% (or 15.0 g / kg) of the weight of the compound functional feed for repairing carbonate-alkali damage in crucian carp, the linoleic acid accounts for 0.7% (or 7.0 g / kg) of the weight of the compound functional feed for repairing carbonate-alkali damage in crucian carp, and the myrcene accounts for 0.5% (or 5.0 g / kg) of the weight of the compound functional feed for repairing carbonate-alkali damage in crucian carp.

[0031] The basic feed includes fishmeal, soybean meal, wheat, and rice bran. The nutritional content of the basic feed is as follows: crude protein ≥29.0%, crude fat ≤12.0%, crude ash ≤15.0%, crude fiber ≥5.0%, moisture ≤12.5%, lysine ≥1.4%, and total phosphorus ≥0.6%.

[0032] The basic feed is a solid particle that can pass through a 60-mesh sieve after being crushed. The compound functional feed for repairing carbonate and alkali damage in crucian carp is a solid particle feed with a particle diameter of 2mm. It is used to feed crucian carp raised in saline-alkali waters. It is used to protect the integrity of the liver tissue structure of crucian carp under saline-alkali stress, so that the liver plates are arranged regularly, the liver cells are uniform, the morphology is normal, and the cell boundaries are clear.

[0033] The compound functional feed for repairing carbonate-alkali damage in crucian carp promotes energy balance, cell growth and development, immune regulation, and inhibits inflammatory factors. It also reduces the difference in gene expression levels of the AMPK signaling pathway in the liver of crucian carp, inhibits protein degradation and excessive apoptosis of hepatocytes.

[0034] Specifically, through setting up an experiment, 180 two-year-old Matsuura silver crucian carp of uniform size, healthy and without external injuries, and with consistent genetic background were selected. Their body length was approximately (13.0±0.5) cm and their weight was approximately (100.10±5.64) g. They were transferred to an indoor temperature-controlled circulating aquaculture equipment, where they were kept under 12-14 hours of light per day and a water temperature of 23-24 ℃. They were raised for 15 days according to the "three fixed points and three patrols" management model (fixed time, fixed location, fixed quantity of feeding, and morning, noon, and evening patrols).

[0035] This experiment included two freshwater control groups (Group C: fed with conventional feed, Group CY: fed with mixed feed), two salt-alkali stress groups: 20 mmol / L NaHCO3 (Group T) and 40 mmol / L NaHCO3 (Group F), and two mixed-feed salt-alkali stress groups (Group TY and Group FY). Each group had three replicates, with 10 fish per replicate. To prevent acute stress in the Matsupu silver carp, the alkalinity was increased to the experimental level at a rate of 10 mmol / L per day, followed by 30 days of exposure. During the experiment, half of the water was changed daily to maintain clean water quality, and ammonia nitrogen was controlled below 2 mg / L. The appropriate alkalinity was prepared using NaHCO3 and calibrated using acid-base titration with 1% methyl orange as an indicator.

[0036] Fish were not fed for 24 hours prior to sampling. They were gently scooped into a basin of water mixed with an anesthetic (MS-222) for deep anesthesia and placed on an ice tray. Blood was collected from the tail vein using a syringe, and the samples were left to stand overnight at 4°C. The samples were then centrifuged at 3000 rpm for 10 minutes at 4°C, and the supernatant was collected and stored at -80°C. Serum samples from groups C, T, F, CY, TY, and FY were collected to measure changes in their biochemical parameters. Liver tissue was immediately removed after blood collection, rinsed with 4°C saline, dried, placed in cryovials, and frozen in liquid nitrogen. After freezing, the tissue was stored at -80°C for later use. Five serum and five liver tissue samples were collected from each group to measure changes in their biochemical parameters. A suitable amount of liver tissue samples were used to establish a liver tissue metabolomics sample processing method. Nine liver tissue samples from each group were used for metabolomics studies, and three liver tissue samples from each group were used for transcriptomics sequencing.

[0037] Fixed liver samples were dehydrated in increments of ethanol, cleared with xylene, embedded in paraffin, sectioned at a thickness of 5 μm, and stained with hematoxylin and eosin (H&E). The stained sections of liver tissue were then observed under an optical microscope and photographed at 400× magnification. The morphology of crucian carp liver under 20 and 40 mmol / L NaHCO3 carbonate alkaline stress and the histological changes of liver under 20 and 40 mmol / L NaHCO3 carbonate alkaline stress by compound functional feed were observed and compared.

[0038] Then, the liver tissue samples stored at -80 ℃ were thawed at 4 ℃. 50 mg was accurately weighed into a 1.5 mL centrifuge tube, and 800 μL of 4 ℃ pre-cooled methanol:water (4:1) and 3 small steel balls were added. The mixture was ground at -20 ℃ at 60 Hz for 3 min, and then allowed to stand at -20 ℃ for 30 min. After centrifugation at 4 ℃ at 13000 rpm for 15 min, 500 μL of the supernatant was taken and filtered through a 0.22 μm organic filter into a sample vial for analysis. During this process, all samples were mixed in equal volumes to prepare QC samples for methodological investigation. QC samples were collected every 10 samples to assess data quality.

[0039] Among them, through Figure 1 It can be seen that there are significant differences in the morphology of crucian carp liver tissue among the freshwater control group, the salt-alkali stress group, and the mixed feed salt-alkali stress group. The liver tissue of crucian carp in groups C and CY is intact, the hepatocytes are uniform, the liver plates are arranged regularly, and the boundaries are clear. The liver cells of crucian carp in group T are cloudy and swollen, the liver sinusoids are dilated, and the liver plates are disordered. The cytoplasm in group F is overflowing (light red) and the cell membrane boundaries are unclear. The liver cells in group TY have clear structure and normal morphology. The liver plates in group FY are arranged neatly and the cell membrane boundaries are obvious.

[0040] Therefore, it can be concluded that compared with group C, the T and F groups showed turbid and swollen hepatocytes, disordered hepatic plates, dilated hepatic sinusoids, cytoplasmic leakage (appearing light red), and unclear cell membrane boundaries, which are closely related to ROS-mediated oxidative stress caused by salt and alkali stress. In contrast, the CY, TY, and FY groups showed intact hepatic tissue structure, normal morphology, neatly arranged hepatic plates, and clear cell membrane boundaries. This indicates that the compound functional feed used to repair carbonate and alkali damage in crucian carp can effectively protect the hepatic tissue structure from oxidative stress damage caused by salt and alkali stress, and maintain the integrity of the hepatic tissue structure under salt and alkali stress, making its hepatic plates neatly arranged, hepatocytes uniform, normal in morphology, and with clear cell boundaries, with no significant difference from the freshwater control group.

[0041] Example 2:

[0042] This invention provides a compound functional feed for repairing carbonate-alkali damage in crucian carp. In the metabolomics analysis, liver tissue extracts from each group of crucian carp were used as samples. The samples were then subjected to non-targeted metabolomics detection using a UPLC-QTOF / MS system to obtain metabolite data. After data preprocessing, the Euclidean distance of metabolite expression levels among the samples was calculated, and hierarchical clustering was used for hierarchical clustering analysis. Figures 2-5 Hierarchical clustering plots showed the changes in DEMs in different treatment groups: C vs. CY, T vs. TY, and F vs. FY. Differences in metabolite levels were visualized using color gradients. To further investigate the effects of mixed diets on the metabolic pathways of crucian carp liver under carbonate-alkali stress, metabolic pathway analysis of DEMs was performed using the MetaboAnalyst 5.0 public metabolomics analysis platform. Based on -log10 (P<0.05) and Impact > 0.01, it was found that the metabolic pathways of glycerophospholipid metabolism, arachidonic acid metabolism, α-linolenic acid metabolism, and sphingolipid metabolism in crucian carp liver were most significantly affected by carbonate-alkali stress.

[0043] In this invention, linoleic acid is made the preferred substrate for liver β-oxidation energy production and an intermediate product of hormone and other signaling molecule biosynthesis. It has multiple physiological functions such as promoting cell growth and development, immune regulation, and inhibiting inflammatory factors. Thus, this invention can reduce the interference of salt-alkali stress through linoleic acid.

[0044] Therefore, it can be concluded that this invention promotes energy balance through fatty acid metabolism pathways, thereby promoting cell growth and development, immune regulation, and inhibiting inflammatory factors, thus alleviating liver inflammation caused by salt-alkali stress and ensuring that the liver is protected from damage under salt-alkali stress.

[0045] Example 3:

[0046] This invention provides a compound functional feed for repairing carbonate-alkali damage in crucian carp, based on... Figure 4 As shown, 1640 DEGs were annotated into three major GO categories: biological processes, cellular components, and molecular functions, and 47 secondary GO categories. The results showed that the BP category was annotated into 19 secondary categories, such as "cellular processes" and "metabolic processes"; the CC category was annotated into 14 secondary categories, such as "cellular parts," "organelles," and "membrane parts"; and the MF category was annotated into 14 secondary categories, such as "adhesion," "transporter activity," and "catalytic activity." To intuitively understand the differences in GO function of differentially expressed genes among different groups, the top 20 GO secondary categories were displayed based on the abundance of DEGs in the GO secondary categories.

[0047] according to Figure 6 As shown, GO enrichment analysis was performed on differentially expressed genes in groups C compared to CY, T compared to TY, and F compared to FY. A P-adjust value < 0.05 was considered a significant GO functional enrichment. The results showed that group C compared to CY had 688 biological processes, 58 cellular components, and 178 molecular functions; group T compared to TY had 32 BP, 1 CC, and 12 MF; and group F compared to FY had 82 BP, 12 CC, and 17 MF. To visually understand the differences in GO functional enrichment among the differentially expressed genes in each group, the top 20 GO-enriched pathways were ranked according to P-adjust significance.

[0048] Compared with the CY group, the C group was significantly enriched in 20 biological processes, including "L-serine biosynthesis", "cellular biogenic amine biosynthesis" and "response to oxygen levels"; compared with the TY group, the T group was significantly enriched in 13 biological processes, including "vitamin catabolic metabolism", "protein maturation regulation" and "protein processing regulation", as well as 7 molecular functions, including "γ-glutamyl cyclotransferase activity" and "heme binding"; compared with the FY group, the F group was significantly enriched in 17 biological processes, including "cholesterol biosynthesis" and "secondary alcohol biosynthesis", 2 molecular functions, "lipid transporter activity" and "nutrient storage activity", and 1 cellular component, "endoplasmic reticulum chaperone complex".

[0049] Compared with the control group, 33 DEGs in the liver of crucian carp under salt-alkali stress were significantly enriched in the AMPK signaling pathway, including 21 differentially expressed genes (DEGs) such as PRKAG1 and PRKAG2, which are involved in encoding AMP-activated protein kinase (AMPK), glucose-6-phosphatase (G6Pase), and 1,6-bisphosphate fructose 1 (FBP1), which were significantly upregulated; and 12 genes such as 6-phosphofructo-2-kinase (PFKFB3), fatty acid synthase (FAS), and acetyl-CoA carboxylase (ACC), which were significantly downregulated.

[0050] Comparative analysis of the control group and the salt-alkali stress compound feed group showed that, except for a significant decrease in HMG-CoA expression level, the expression levels of the other genes mentioned above in crucian carp liver did not differ significantly from those in the control group (P-value > 0.05). Comparative analysis of the salt-alkali stress group and the salt-alkali stress compound feed group showed that the expression levels of three genes encoding insulin receptor substrate (IRS1), insulin receptor substrate (IRS2), and 3-phosphoinositol-dependent protein kinase 1 (PDPK-1) were significantly decreased, while the expression level of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) was significantly increased. This indicates that the present invention can significantly reduce the differences in AMPK signaling pathway gene expression levels in crucian carp liver caused by salt-alkali stress.

[0051] Therefore, it can be concluded that the AMPK signaling pathway plays a central role in maintaining cellular energy homeostasis by regulating glucose and lipid metabolism.

[0052] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A compound functional feed for repairing carbonate-alkali damage in crucian carp, characterized in that: The compound functional feed for repairing carbonate-alkali damage in crucian carp consists of α-ketoglutarate, linoleic acid, myrcene, and a basic feed. The α-ketoglutaric acid accounts for 1.5% of the weight of the compound functional feed used to repair carbonate-alkali damage in crucian carp. The linoleic acid accounts for 0.7% of the weight of the compound functional feed used to repair carbonate-alkali damage in crucian carp. The myrcene accounts for 0.5% of the weight of the compound functional feed used to repair carbonate-alkali damage in crucian carp.

2. The compound functional feed for repairing carbonate-alkali damage in crucian carp according to claim 1, characterized in that: The basic feed includes fishmeal, soybean meal, wheat, and rice bran.

3. The compound functional feed for repairing carbonate-alkali damage in crucian carp according to claim 2, characterized in that: The nutritional composition of the basic feed is as follows: crude protein ≥29.0%, crude fat ≤12.0%, crude ash ≤15.0%, crude fiber ≥5.0%, moisture ≤12.5%, lysine ≥1.4%, and total phosphorus ≥0.6%.

4. The compound functional feed for repairing carbonate-alkali damage in crucian carp according to claim 3, characterized in that: The basic feed consists of solid particles that can be pulverized and pass through a 60-mesh sieve.

5. The compound functional feed for repairing carbonate-alkali damage in crucian carp according to claim 4, characterized in that: The compound functional feed used to repair carbonate-alkali damage in crucian carp is a solid pellet feed with a pellet diameter of 2 mm.

6. The compound functional feed for repairing carbonate-alkali damage in crucian carp according to claim 5, characterized in that: The compound functional feed for repairing carbonate and alkali damage in crucian carp is used to feed crucian carp raised in saline-alkali waters.

7. The compound functional feed for repairing carbonate-alkali damage in crucian carp according to claim 6, characterized in that: The compound functional feed used to repair carbonate and alkali damage in crucian carp is designed to protect the integrity of the liver tissue structure of crucian carp under salt and alkali stress, ensuring that the liver plates are arranged regularly, the liver cells are uniform, the morphology is normal, and the cell boundaries are clear.

8. The compound functional feed for repairing carbonate-alkali damage in crucian carp according to claim 7, characterized in that: The compound functional feed used to repair carbonate-alkali damage in crucian carp promotes energy balance, cell growth and development, immune regulation, and inhibits inflammatory factors.

9. The compound functional feed for repairing carbonate-alkali damage in crucian carp according to claim 8, characterized in that: The compound functional feed for repairing carbonate-alkali damage in crucian carp is used to reduce the difference in gene expression levels of the AMPK signaling pathway in the liver of crucian carp, and to inhibit protein degradation and excessive apoptosis of hepatocytes.

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