Functional feed for promoting hepatopancreas health of leuciscus waleckii and application

Abstract

The invention belongs to the technical field of fish feed, and particularly relates to functional feed for promoting hepatopancreas of leuciscus waleckii to be healthy and application. The functional feed for promoting hepatopancreas health of the leuciscus waleckii comprises a basal feed and an additive chlorogenic acid, the addition amount of the chlorogenic acid is 0.02 to 0.08 percent. The functional feed capable of promoting hepatopancreas health of the leuciscus waleckii is simple in component, easy in raw material obtaining, safe and harmless, and by adding a proper amount of chlorogenic acid, the fish growth and feed utilization rate can be effectively improved, the lipid metabolism function, the immune function and the oxidation resistance are improved, and the growth performance and hepatopancreas lipolysis of the leuciscus waleckii are effectively promoted.

Description

Technical Field

[0001] The invention belongs to the technical field of fish feed, and in particular relates to a functional feed for promoting the hepatopancreas health of Leuciscus walleye and application thereof. Background Art

[0002] In the context of antibiotic substitution and the pursuit of high-quality aquatic products, it is very important to explore a green, pollution-free, natural and pollution-free plant extract as a feed additive. Chlorogenic acid (CGA), as a natural plant extract of Chinese herbal medicine, has attracted widespread attention from scholars at home and abroad for its application in aquatic feed. CGA is a polyphenol substance that is widely distributed in fruits, vegetables, and Chinese herbal medicines, such as apples, carrots, honeysuckle, and eucommia. CGA has multiple pharmacological functions, such as anti-inflammatory, antioxidant, and lipid metabolism regulation.

[0003] Leuciscus waleckii belongs to the genus Leuciscus, subfamily Leuciscinae, family Cyprinidae, and is widely distributed in Europe, Siberia, the Caucasus, and the Heilongjiang River Basin in China. Leuciscus waleckii is popular among consumers for its delicious taste, tender meat, high protein content, essential amino acids such as methionine and lysine, unsaturated fatty acids, vitamin A and vitamin D. It has a wide temperature range and is suitable for high-density pond farming. It is a valuable economic fish popular in the market and was rated as one of the top ten characteristic aquatic germplasm resources in my country in 2022.

[0004] Aquatic feed is an important material basis for the development of aquaculture. However, modern large-scale intensive aquaculture generally faces problems such as high stocking density, large feed input and residual amount, which often cause large-scale epidemic diseases and lead to reduced production performance of aquatic animals. And with the irrational addition of antibiotics in aquatic feed, drug residues and drug resistance have frequently appeared. Therefore, as my country's aquaculture continues to transform and upgrade towards green and healthy aquaculture, the research and development of environmentally friendly functional high-activity aquatic feed and scientific and reasonable "antibiotic replacement" ecological aquaculture model have become hot topics today.

[0005] In view of this, the present invention is proposed. Summary of the invention

[0006] The purpose of the present invention is to provide a functional feed and application for promoting the hepatopancreas health of Leuciscus walleye; the functional feed can effectively improve the growth and feed utilization rate of fish, lipid metabolism function, immune function and antioxidant capacity by adding an appropriate amount of chlorogenic acid, and can effectively promote the growth performance of Leuciscus walleye and the decomposition of hepatopancreas fat.

[0007] In order to overcome the deficiencies of the prior art, the present invention provides the following technical solutions:

[0008] A functional feed for promoting the hepatopancreas health of Leuciscus walleye, comprising a basic feed and an additive chlorogenic acid; the added amount of the chlorogenic acid is 0.02-0.08% (e.g. 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%).

[0009] Based on the above scheme, as a preferred method, the amount of chlorogenic acid added is 0.04%. The present invention has been confirmed through a large number of studies that when the amount of chlorogenic acid added is 0.04%, the growth performance and feed efficiency of Walleye fish can be improved, and through multiple pathways such as enzyme activity and gene regulation, hepatopancreatic fat deposition can be inhibited, fat decomposition can be promoted to prevent lipid peroxidation in the fish body, the NF-κB signaling pathway can be activated to reduce the occurrence of inflammatory response, and the Keap1-Nrf2 signaling pathway can be activated to improve antioxidant capacity.

[0010] On the basis of the above scheme, as a preference, the basic feed comprises the following raw materials in parts by weight: 29.5-30.5 parts of fish meal, 11-12 parts of peeled soybean meal, 9.5-10.5 parts of concentrated soy protein, 4.5-5.5 parts of corn gluten meal, 17.5-18.5 parts of flour, 2-3 parts of fish oil, 2-3 parts of soybean oil, 0.4-0.6 parts of mineral and vitamin premix, 1.5-2.5 parts of monocalcium phosphate, 0.2-0.4 parts of choline chloride, 0.07-0.09 parts of vitamin C ester, 2.5-3.5 parts of microcrystalline cellulose and 14-15 parts of dextrin.

[0011] The weight parts of the present invention mainly include the disclosed numerical range, any numerical value (including integer and decimal) or any interval of two numerical values ​​in the disclosed range, or multiple discontinuous intervals, and also include numerical values ​​or numerical ranges close to the end value of the numerical range whose effects can be expected to be similar, such as 5-10 parts, not only including 5 parts, 6 parts, 7 parts, 8 parts, 9 parts, 10 parts and any interval of two parts, for other numerical ranges, not exemplified one by one, all are included in the present invention. Therefore, the present invention also includes the sub-range of any numerical range that has been directly disclosed or any specific numerical value therein.

[0012] In order to further optimize the effect of the functional feed, the present invention studies the influence of different proportions of various components of the functional feed on its effect, and obtains the optimal component ratios respectively. The functional feed is made of the following raw materials, calculated by mass percentage: 30% fish meal, 11.53% peeled soybean meal, 10% soybean protein concentrate, 5% corn protein powder, 18% flour, 2.44% fish oil, 2.44% soybean oil, 0.04% chlorogenic acid, 0.5% mineral and vitamin premix, 2% monocalcium phosphate, 0.3% choline chloride, 0.08% vitamin C ester, 3.03% microcrystalline cellulose and 14.64% dextrin.

[0013] On the basis of the above scheme, as a preferred embodiment, in the basic feed, the crude protein content is 36-37%, the crude fat content is 7-8%, and the crude ash content is 6-7%. The higher the feed protein level, the faster the fish will grow. There are also differences in the protein content requirements of fish with different feeding habits in the feeding composition. Omnivorous and herbivorous fish have relatively low requirements for protein, and the demand is between 22% and 45%. The higher the feed protein level, the higher or lower it will affect the growth performance, immune function and metabolic function of fish. After research, it was found that the theoretical value of crude protein content is 36-37%, which is the appropriate range for promoting growth. After that, the actual measured value in the feed was within this range through national standard measurement (Kjeldahl nitrogen determination method), so this numerical range was obtained. Crude fat and crude ash are like crude protein.

[0014] In addition, the present invention also provides a method for preparing the functional feed as described above, comprising the following steps:

[0015] S1. The raw materials of chlorogenic acid and basic feed are crushed and sieved respectively, and then mixed evenly; then put into a screw extruder and extruded into granular feed;

[0016] S2. The granular feed obtained in step S1 is dried, sealed, and frozen for storage to obtain the functional feed.

[0017] On the basis of the above scheme, preferably, in step S1, the mesh size of the sieving is 0.2-0.3 mm; and / or the particle diameter of the granular feed is 1.5 mm.

[0018] In step S2, the drying condition is: drying at 45°C for 24 hours; and / or the freezing storage temperature is -20°C.

[0019] In addition, the present invention also provides the application of the functional feed in the field of fish farming. It should be noted that the functional feed provided by the present invention is not only suitable for the breeding of Leuciscus vachelli, but also can be applied to other fish, including but not limited to carp, sea bass, catfish, rainbow trout, loach, etc.

[0020] Preferably, the application includes promoting fish growth performance, lipid metabolism function, immune function and antioxidant capacity.

[0021] Compared with the prior art, the technical solution of the present invention has at least the following technical effects:

[0022] (1) The functional feed for promoting the hepatopancreas health of the present invention is simple in components, the raw materials are easily available, and it is safe and harmless. By adding an appropriate amount of chlorogenic acid, it can effectively improve the growth and feed utilization rate of fish, lipid metabolism function, immune function and antioxidant capacity, and effectively promote the growth performance of the fish and the decomposition of hepatopancreas fat.

[0023] (2) The present invention evaluates the effects of adding different levels of CGA on the growth performance, lipid metabolism function, immune function, and antioxidant capacity of Leuciscus walleye, in order to provide a scientific basis for the development and application of green alternative antibiotic feed additives and provide a theoretical reference for improving fish growth and health.

[0024] (3) The method for preparing the functional feed for promoting the hepatopancreas health of Leuciscus walleye of the present invention has simple steps, low cost and is suitable for practical use. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The drawings constituting a part of the present application are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. Among them:

[0026] Figure 1 This is the lipid metabolism and fat deposition diagram of the hepatopancreas of Leuciscus walleye in the CK group without CGA addition;

[0027] Figure 2 This is the lipid metabolism and fat deposition diagram of the hepatopancreas of the C1 group supplemented with CGA;

[0028] Figure 3 This is the lipid metabolism and fat deposition diagram of the hepatopancreas of Leuciscus walleye in the C2 group supplemented with CGA;

[0029] Figure 4 This is the lipid metabolism and fat deposition diagram of the hepatopancreas of Leuciscus walleye in the C3 group supplemented with CGA;

[0030] Figure 5 This is the lipid metabolism and fat deposition diagram of the hepatopancreas of the C4 group supplemented with CGA;

[0031] Figure 6 It is a bar graph showing the effects of different CGA levels on the ACC-1 mRNA level in the hepatopancreas of Leuciscus walleye;

[0032] Figure 7It is a bar graph showing the effect of different CGA levels on the PPAR-γ mRNA level in the hepatopancreas of Leuciscus walleye;

[0033] Figure 8 It is a bar graph showing the effects of different CGA levels on the FAS mRNA level in the hepatopancreas of Leuciscus walleye;

[0034] Fig. 9 It is a bar graph showing the effect of different CGA levels on the mRNA level of PPAR-β mRNA in the hepatopancreas of Leuciscus walleye;

[0035] Fig.10 It is a bar graph showing the effects of different CGA levels on the level of MyD88 mRNA related to hepatopancreatic inflammation in Leuciscus walleye;

[0036] Fig.11 It is a bar graph showing the effects of different CGA levels on the mRNA level of NF-κBp65 related to hepatopancreatic inflammation in Leuciscus walleye;

[0037] Fig.12 It is a bar graph showing the effects of different CGA levels on the level of TGF-β mRNA related to hepatopancreatic inflammation in Leuciscus walleye;

[0038] Fig.13 It is a bar graph showing the effect of different CGA levels on the level of TNF-α mRNA related to hepatopancreatic inflammation in Leuciscus walleye;

[0039] Fig.14 The bar graph shows the effect of different CGA levels on the mRNA level of antioxidant Nrf2 in the hepatopancreas of Leuciscus walleye;

[0040] Fig.15 It is a bar graph showing the effect of different CGA levels on the level of antioxidant Keap-1 mRNA in the hepatopancreas of Leuciscus walleye;

[0041] Fig.16 It is a bar graph showing the effect of different CGA levels on the antioxidant CAT mRNA level in the hepatopancreas of Leuciscus walleye;

[0042] Fig.17 This is a bar graph showing the effects of different CGA levels on the antioxidant HO-1 mRNA level in the hepatopancreas of Leuciscus walleye. DETAILED DESCRIPTION

[0043] In order to make the purpose, technical scheme and advantages of the present invention clearer, the technical scheme in the embodiment of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. It should be understood by those skilled in the art that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without making creative work are within the scope of protection of the present invention. The process parameters of the following embodiments that do not specify specific conditions are usually based on conventional conditions.

[0044] The endpoints and any values ​​of the ranges disclosed in the present invention are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be regarded as specifically disclosed in the present invention.

[0045] In the embodiments of the present invention, the experimental methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used are all commercially available unless otherwise specified.

[0046] Example 1

[0047] 1. Materials and methods

[0048] 1.1 Preparation of test materials

[0049] Fish meal, dehulled soybean meal, soybean protein concentrate, corn gluten meal, flour, fish oil, soybean oil, mineral and vitamin premix (mg / kg premix, purchased from Boeing Feed Co., Ltd., Shenyang, China: vitamin A 960000IU; vitamin D 560000IU; vitamin E 24000 mg; vitamin K 1620 mg; niacinamide 4580 mg; pantothenic acid 7020 mg; folic acid 1060 mg; biotin 40 mg; inositol 30200 mg; copper 961 mg; Iron 40600 mg; manganese 5520 mg; cobalt 64 mg; iodine 134 mg; zinc 23200 mg; selenium 84 mg), monocalcium phosphate, choline chloride, vitamin C ester, microcrystalline cellulose and dextrin are used as basic feed raw materials, and the added amount of chlorogenic acid is: 0.00% (corresponding to CK group), 0.02% (corresponding to C1 group), 0.04% (corresponding to C2 group), 0.06% (corresponding to C3 group) and 0.08% (corresponding to C4 group), respectively, to configure five functional feeds.

[0050] The preparation method is as follows: After the feed raw materials are crushed, they are passed through a 0.246 mm sieve, weighed according to the feed formula in Table 1, mixed evenly, and made into 1.5 mm diameter particles using a multifunctional screw extruder (YL90L4, Jilin Agricultural University, Changchun). The feed particles are dried in an oven at 45 °C for 24 h, sealed in a sealed bag, and then stored in a -20 °C refrigerator for later use.

[0051] Table 1 Feed formula and main nutrients (%, air-dried basis)

[0052]

[0053] 1.2 Feeding experiment

[0054] The Walleye fish used in the experiment were purchased from Yongshun Fishery, Xingling Township, Meihekou City, Jilin Province. This experiment was designed as a single factor factorial experiment. 600 Walleye fish were randomly divided into 5 groups, with 3 replicates in each group, and 40 Walleye fish were released in each replicate for an 8-week feeding experiment. Before the formal experiment began, they were temporarily raised for 2 weeks. During the temporary raising period, the CK group feed was fed and aeration was maintained for 24 hours. During the feeding period, artificial feeding was used, and feeding was carried out 3 times a day (7:00 am, 12:00 pm, and 17:00 pm), and the feeding rate was 3% to 5% of the body weight of Walleye fish. During the breeding period, the dissolved oxygen was kept>5mg / L, the water temperature was between 24℃ and 28℃, the pH was between 7.5 and 8.5, the ammonia nitrogen was <0.3mg / L, and the nitrite was <0.05mg / L. The water was changed once every 2 days, and 1 / 3 to 1 / 4 of the total water volume was changed each time.

[0055] 1.3 Sample collection

[0056] After the feeding experiment, the fish were stopped from feeding for 24 h, anesthetized with 100 mg / L MS-222 (3-aminobenzoic acid ethyl ester, Sigma, USA), and the group weight of each tank was weighed. Eight fish were randomly selected from each tank, and their body weight, body length, visceral weight, and hepatopancreas weight were measured for the calculation of growth indicators.

[0057] Fifteen Leuciscus walleye were randomly selected from each tank, and blood was collected from the tail vein using a syringe. The samples were allowed to stand at 4°C for 24 hours and then centrifuged at 3000g for 10 minutes at 4°C. The supernatant was collected and stored at -80°C for analysis of lipid metabolism, immunity and antioxidant indicators. Muscles above the lateral line and below the dorsal fin were collected for the determination of muscle nutrient components. The liver and pancreas were collected, quickly frozen in liquid nitrogen, and stored at -80°C for the determination of lipid metabolism enzyme activity, antioxidant capacity, and gene mRNA expression.

[0058] Ten Leuciscus walleye were randomly selected from each tank, and the hepatopancreas of the same size and position were collected and preserved in paraformaldehyde for observation with oil red staining.

[0059] 1.4 Sample determination

[0060] 1.4.1 Determination of growth indicators and muscle nutrients

[0061] Growth performance and feed utilization indices were calculated according to the following formula:

[0062] Mass gain rate (WGR) = (Wt-W0) / W0×100%…………………………(1)

[0063] Specific growth rate (SGR) = (lnWt - lnW0) / t × 100% (2)

[0064] Feed conversion ratio (FCR) = I t / (Wt-W0)……………………(3)

[0065] Feed efficiency (FER) = (Wt - W0) / I t ×100%…………………………(4)

[0066] Protein efficiency (PER) = (Wt - W0) / (I t ×P)×100%………………………(5)

[0067] Liver to body ratio (HIS) = W H / Wt×100%……………………(6)

[0068] Visceral-to-body ratio (VSI) = W V / Wt×100%……………(7)

[0069] Fullness (CF) = Wt / L 3 ×100……………………(8)

[0070] Survival rate (SR) = final number / initial number × 100%…………………(9)

[0071] Where: Wt, W0 are the final and initial fish weights (g), t is the test time (d); I t is the mass of dry feed ingested (g); P is the crude protein content of feed (%); L is the length of fish (cm); W H W is the mass of hepatopancreas (g); V is the visceral mass (g).

[0072] The nutrient composition of feed and muscle was determined using the standard method published by AOAC. The specific operating steps were based on the method described by Li et al., that is, crude protein was determined by the Kjeldahl method; crude fat was determined by the Soxhlet extraction method; crude ash was determined by the 550°C muffle furnace ignition method; and moisture was determined by the 105°C constant temperature drying weight loss method.

[0073] 1.4.2 Enzyme activity assay

[0074] Nanjing Jiancheng kit was used to determine the levels of high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), triglycerides (TG) and total cholesterol (TC) in serum, and acetyl-CoA carboxylase (ACC), adipose triglyceride lipase (ATGL) and fatty acid synthase (FAS) in hepatopancreas of Leuciscus wallichii.

[0075] Nanjing Jiancheng kit was used to determine the lysozyme (LZM) and alkaline phosphatase (AKP) in the serum of Leuciscus walleye, and ELISA kits (purchased from Jingmei Bioengineering Co., Ltd.) were used to determine the levels of immunoglobulin M (IgM), complement 3 (C3), and complement 4 (C4) in the serum.

[0076] Nanjing Jiancheng kit was used to determine the total antioxidant capacity (T-AOC), catalase (CAT), superoxide dismutase (T-SOD), glutathione peroxidase (GSH-Px) activities and malondialdehyde (MDA) content in the serum and hepatopancreas of Leuciscus walleye.

[0077] 1.4.3 Real-time quantitative PCR analysis

[0078] Total RNA was extracted from the intestine of carp using tissue lysis buffer (AC0101-B, Si Ke Jie), chloroform and isopropanol. Subsequently, the concentration and quality of total RNA were evaluated using a UV-visible spectrophotometer (NanoDrop2000, Thermo, USA) and gel electrophoresis, respectively. After ensuring that the total RNA met the specified standards, it was reverse transcribed into cDNA using a reverse transcription kit (AG0304-B, Si Ke Jie). Table 2 provides primer information, and β-actin was selected as the housekeeping gene. Fluorescence quantitative PCR used the TB Green dye method, and the reaction system was 20 μL, including 10 μL TransStart Green qPCR SuperMix (2×), 1 μL upstream primer, 1 μL downstream primer, 1 μL cDNA template, 7 μL nuclease-free water, and was performed in the ABI (7500) RT-PCR system. All samples were tested three times. After the qRT-PCR reaction was completed, 2 -ΔΔCT Methods The relative expression levels of all target gene mRNAs were calculated.

[0079] Table 2 Gene primer sequences

[0080]

[0081]

[0082] 1.4.4 Observation of hepatopancreas oil red stained sections

[0083] Lipid accumulation in the hepatopancreas of Leuciscus walleye was observed by staining with Oil Red O. The hepatopancreas samples were fixed in tissue fixative and then frozen, treated with 200 g / L and 300 g / L sucrose buffer for 24 h, embedded in paraffin and cut into thin sections, stained with 60% Oil Red O staining solution, and finally observed and imaged with a fluorescent inverted microscope.

[0084] 1.5 Statistical analysis

[0085] Data were expressed as (mean ± standard deviation) and SPSS26 software was used to perform one-way ANOVA (LSD). If the variance analysis of each group was significant, Duncan's multiple comparison test was used for multiple comparisons and orthogonal polynomial analysis was performed. The significance level was set at P < 0.05.

[0086] 2 Test Results

[0087] 2.1 Effects of different levels of chlorogenic acid in feed on growth performance and muscle nutritional components of Leuciscus walleye

[0088] As shown in Table 3, the FBW (final body weight), WGR (weight gain rate), SGR (specific growth rate), FER (feed efficiency), PER (protein efficiency) and CF (fatness) of Leucissima walleye were significantly quadratically correlated with the CGA supplementation level (P<0.05). Compared with the CK group, the FBW, WGR and SGR of the C1-C4 groups were significantly increased (P<0.05); compared with the CK group, the CF, FER and PER of the C2 and C3 groups were significantly increased (P<0.05), and there was no significant effect on the CF, FER and PER of the C1 and C4 groups (P>0.05); compared with the CK group, there was no significant effect on the VSI and HSI of the C1-C4 groups (P>0.05).

[0089] Table 3 Effects of different levels of chlorogenic acid in feed on growth performance and feed efficiency of Leuciscus walleye

[0090]

[0091]

[0092] As shown in Table 4, the crude protein content in the muscle of Leuciscus walleye showed a significant quadratic relationship with the increase of CGA addition level (P<0.05). Compared with the CK group, the crude protein content of groups C1 to C4 was significantly increased (P<0.05). There was no significant effect on crude fat, crude ash and moisture among the groups (P>0.05).

[0093] Table 4 Effects of different levels of chlorogenic acid in feed on the nutritional components of muscle of Leuciscus walleye

[0094]

[0095]

[0096] 2.2 Effects of different levels of chlorogenic acid in feed on the number of lipid droplets, lipid metabolism function and related gene expression in the hepatopancreas of Leuciscus walleye

[0097] like Figure 1-5 As shown, compared with the CK group, the addition of CGA significantly reduced the number of lipid droplets in the hepatopancreas of Leuciscus walleye.

[0098] As shown in Table 5, the activities of HDL-C, LDL-C, TC and TG in the serum of Leuciscus walleye were significantly linearly correlated with the level of CGA addition (P<0.05). Compared with the CK group, the activities of HDL-C in the serum of the C1-C4 groups increased significantly (P<0.05), and the activities of LDL-C, TC and TG decreased significantly (P<0.05).

[0099] Table 5 Effects of different levels of chlorogenic acid in feed on the activities of serum lipid metabolism-related enzymes in Leuciscus walleye

[0100]

[0101]

[0102] As shown in Table 6, the activities of ACC, ATGL, FAS and TG in the hepatopancreas of Leuciscus walleye showed a significant quadratic relationship with the level of CGA addition (P<0.05). Compared with the CK group, the activities of ACC, FAS and TG in the hepatopancreas of the C1-C4 groups decreased significantly (P<0.05), and the activity of ATGL increased significantly (P<0.05).

[0103] Table 6 Effects of different levels of chlorogenic acid in feed on the activities of lipid metabolism enzymes in the hepatopancreas of Leuciscus walleye

[0104]

[0105]

[0106] like Figure 6-9As shown in the results, the mRNA levels of ACC-1, PPAR-γ, FAS and PPAR-β in the hepatopancreas of Leucis vachelli showed a significant quadratic relationship with the CGA supplementation level (P<0.05). Compared with the CK group, the mRNA levels of PPAR-β in the hepatopancreas of groups C1 to C4 were significantly increased (P<0.05), and the mRNA levels of FAS and ACC-1 were significantly decreased (P<0.05); compared with the CK group, the mRNA levels of PPAR-γ in the hepatopancreas of groups C2 and C3 were significantly decreased (P<0.05), and there was no significant effect on the mRNA levels of PPAR-γ in the hepatopancreas of groups C1 and C4 (P>0.05).

[0107] 2.3 Effects of different levels of CGA in feed on immune function and expression of inflammation-related genes in Leuciscus walleye

[0108] As shown in Table 7, the serum LZM, AKP, IgM, C3 and C4 levels of Leucocephalus walleye showed a significant quadratic relationship with the chlorogenic acid supplementation level (P<0.05). Compared with the CK group, the serum LZM, IgM and C4 activities of groups C1 to C4 increased significantly (P<0.05); the serum AKP activity of group C2 increased significantly (P<0.05); compared with the CK group, the serum C3 activity of groups C2 and C3 increased significantly (P<0.05), the serum C3 activity of group C4 decreased significantly (P<0.05), and the serum C3 activity of group C1 had no significant effect (P>0.05).

[0109] Table 7 Effects of different levels of chlorogenic acid in feed on the activities of serum immune-related enzymes in Leuciscus walleye

[0110]

[0111]

[0112] Figure 10-13 As shown in the results, the mRNA levels of MyD88, NF-κBp65, TGF-β and TNF-α in the hepatopancreas of Leucissima walleye showed a significant quadratic relationship with the CGA supplementation level (P<0.05). Compared with the CK group, the mRNA levels of MyD88 and TNF-α in the hepatopancreas of groups C1 to C4 were significantly decreased (P<0.05), and the mRNA level of TGF-β was significantly increased (P<0.05); compared with the CK group, the mRNA level of NF-κBp65 in the hepatopancreas of group C2 was significantly decreased (P<0.05), and the mRNA level of NF-κBp65 in the hepatopancreas of groups C1, C3 and C4 was not significantly affected (P>0.05).

[0113] 2.4 Effects of different levels of CGA in feed on antioxidant capacity and related gene expression in Leuciscus walleye

[0114] As shown in Table 8, T-AOC, MDA, T-SOD, CAT and GSH-Px in the serum of Leuciscus walleye showed a significant quadratic relationship with the level of CGA addition (P<0.05). Compared with the CK group, the activities of T-SOD, CAT and GSH-Px in groups C1 to C4 increased significantly (P<0.05), and the content of MDA decreased significantly (P<0.05); compared with the CK group, the serum T-AOC levels in groups C2 and C3 increased significantly (P<0.05), and there was no significant effect on the serum T-AOC levels in groups C1 and C4 (P>0.05).

[0115] As shown in Table 9, T-AOC, MDA, T-SOD, CAT and GSH-Px in the hepatopancreas of Leucissima walleye showed a significant quadratic relationship with the CGA addition level (P<0.05); compared with the CK group, the CAT and GSH-Px activities of groups C1 to C4 were significantly increased (P<0.05), and the MDA content was significantly decreased (P<0.05); compared with the CK group, the hepatopancreas T-SOD and T-AOC activities of groups C1 to C3 were significantly increased (P<0.05), and there was no significant effect on the serum hepatopancreas T-SOD and T-AOC activities of group C4 (P>0.05).

[0116] Table 8 Effects of different levels of chlorogenic acid on the activities of serum antioxidant-related enzymes in Leuciscus walleye

[0117]

[0118] Table 9 Effects of different levels of chlorogenic acid in feed on antioxidant enzyme activities in the hepatopancreas of Leuciscus walleye

[0119]

[0120]

[0121] from Figure 14-17 As shown in the results, Nrf2, Keap-1, HO-1 and CAT in the hepatopancreas of Leucissima walleye showed a significant quadratic relationship with the level of CGA addition (P<0.05). Compared with the CK group, the mRNA levels of Nrf2 and CAT in the hepatopancreas of groups C1 to C4 were significantly increased (P<0.05); compared with the CK group, the mRNA level of HO-1 in the hepatopancreas of groups C1 to C3 was significantly increased (P<0.05), and the mRNA level of HO-1 in the hepatopancreas of group C4 was not significantly affected (P>0.05); compared with the CK group, the mRNA level of Keap-1 in the hepatopancreas of groups C1 to C3 was significantly decreased (P<0.05), and the mRNA level of Keap-1 in the hepatopancreas of group C4 was not significantly affected (P>0.05).

[0122] 3. Evaluation test results

[0123] 3.1 Effects of different levels of chlorogenic acid in feed on growth performance and muscle nutrients of Leuciscus walleye

[0124] This experimental study found that the addition of 0.04% to 0.06% CGA in the feed significantly increased the FBW, WGR, SGR, CF, FER and PER of Walleye fish, indicating that the addition of CGA in the feed can promote the growth performance of aquatic animals and improve feed efficiency. This may be because CGA has strong antioxidant, anti-inflammatory and lipid-lowering effects, which is beneficial to maintain and improve the health of aquatic animals, thereby improving the growth performance of aquatic animals. Aquatic animals are an important part of the human diet and provide humans with rich protein. This study showed that the addition of CGA to the feed significantly increased the crude protein content in the muscle of Walleye fish, indicating that the addition of CGA can improve the nutritional value of fish muscle and enhance muscle quality.

[0125] 3.2 Effects of different levels of chlorogenic acid in feed on lipid metabolism in Yarrowia walleyeii

[0126] The liver is an important organ in the lipid metabolism process of aquatic animals. Increased serum TC, TG and LDL-C levels are considered as risk indicators of lipid accumulation and lipid metabolism disorders. In this experiment, the HDL-C activity in the serum of Walleye fish increased significantly after adding 0.04% CGA to the feed, while the LDL-C, TG and TC activities decreased significantly, indicating that the addition of 0.04% CGA to the feed can reduce the serum fat level of Walleye fish. It shows that the addition of CGA to the feed can promote fat decomposition and inhibit lipid deposition in aquatic animals. ATGL can catalyze the hydrolysis of TG, thereby affecting fat decomposition. ACC is the key rate-limiting enzyme in fatty acid synthesis. FAS also plays an important role in fat synthesis, and its activity is regulated by ACC. PPAR-β improves lipid utilization by regulating the expression of target genes responsible for encoding β-oxidation enzymes and inflammatory functions. PPAR-γ is the main regulator of adipocyte differentiation, and its activation is related to the expression of many important genes affecting energy metabolism. The results of this experiment showed that with the increase of CGA levels, the activities of ACC and FAS in the hepatopancreas of Walleye showed a downward trend, and the activity of ATGL increased significantly. When 0.04% CGA was added, the levels of ACC-1, FAS and PPAR-γ mRNA in the hepatopancreas decreased significantly, and PPAR-β mRNA increased significantly. This shows that adding 0.04% CGA to the feed can promote fat decomposition, inhibit fat synthesis, and reduce fat deposition in the hepatopancreas of Walleye. In addition, the results of this experiment found that the TG level in the hepatopancreas of Walleye was consistent with the TG level in the serum, which shows that adding 0.04% CGA to the feed can inhibit fat deposition in the hepatopancreas of Walleye, and this result is consistent with the fat content of the hepatopancreas stained with oil red in this experiment.

[0127] 3.3 Effects of different levels of chlorogenic acid in feed on the immune function of Leuciscus walleye

[0128] Normal immune function of fish is necessary to maintain its healthy growth. AKP plays an important role in the body's removal of foreign matter and resistance to pathogen infection. LZM can remove Gram-positive and Gram-negative bacteria by destroying bacterial cell walls. C3 and C4 are key members of the complement system, and immune function can be improved by activating the complement system. Antigen stimulation induces the production of immunoglobulins, and IgM is an important immunoglobulin in fish. The results of this experiment show that the addition of 0.04% CGA to the feed significantly increases the levels of AKP, LZM, C3, C4 and IgM in the serum of Walleye fish, indicating that the appropriate level of CGA has a promoting effect on the serum immune function of Walleye fish and improves the body's specific immune function. The present invention studies the NF-κB signaling pathway, which is closely related to the inflammatory response. The NF-κB family exists in organisms and regulates the expression of many genes involved in inflammation as a transcription factor. MyD88 overexpression can lead to spontaneous activation of NF-κB. TNF-α is a proinflammatory cytokine, while TGF-β is an anti-inflammatory cytokine, both of which play an important role in immune regulation. The results of this experiment showed that the addition of 0.04% CGA in feed significantly downregulated the levels of NF-κBp65, MyD88 and TNF-α mRNA in the hepatopancreas, and upregulated the level of TGF-β mRNA, indicating that CGA can activate the animal's cellular immunity and humoral immunity through a series of anti-inflammatory reactions such as inhibiting immune cells and immune factors and activating immunosuppressive factors, thereby enhancing the animal's immune function.

[0129] 3.4 Effects of different levels of chlorogenic acid in feed on the antioxidant capacity of Leuciscus walleye

[0130] Antioxidant capacity is an important parameter to measure the health status of the body. MDA is a key marker for evaluating oxidative damage in the body. The level of T-AOC can reflect the total antioxidant capacity level composed of antioxidant substances and antioxidant enzymes in the body; T-SOD can convert reactive oxygen such as peroxides and free radicals in the body into H2O2, and CAT can catalyze H2O2 to produce water and oxygen; GSH-Px can convert peroxides into hydroxyl compounds, thereby protecting the body from oxidative damage. The results of this experiment showed that the serum MDA level of Walleye fish significantly decreased when 0.04% to 0.06% CGA was added to the feed, and the levels of CAT, T-AOC, T-SOD and GSH-Px increased significantly, indicating that the appropriate level of CGA can improve the antioxidant capacity of Walleye fish. The Keap1-Nrf2 signaling pathway is a key endogenous antioxidant pathway in animals. Under oxidative stress, the production of ROS surges, promoting the dissociation of Nrf2 from Keap1. Subsequently, Nrf2 translocates to the nucleus and regulates the expression of antioxidant genes, thereby counteracting oxidative stress. HO-1 is a key target gene of Nrf2. It can be rapidly activated during oxidative stress and inhibit the occurrence of oxidative stress response, playing a protective role. CAT is a key gene for enzymatic antioxidant defense in animals, responsible for neutralizing excessive ROS. The results of this experiment showed that the addition of 0.02% to 0.06% CGA to the feed significantly decreased the MDA level in the hepatopancreas of Walleye, while CAT, T-AOC, T-SOD and GSH-Px increased significantly. In addition, the addition of 0.04% CGA to the feed significantly increased the mRNA expression levels of Nrf2, HO-1 and CAT in the hepatopancreas of Walleye, and downregulated the mRNA expression level of Keap1. This shows that the addition of CGA to the feed activated the Keap1-Nrf2 signaling pathway, regulated the expression of antioxidant-related genes, and thus enhanced the body's antioxidant capacity. CGA can enhance the level of T-AOC and SOD activity in the hepatopancreas, reduce the content of MDA, and upregulate the expression levels of Nrf2, SOD, CAT, GPX and HO-1 mRNA, reduce the occurrence of oxidative stress, and enhance the body's antioxidant capacity. This may be due to the special molecular structure of CGA containing 5 active hydroxyl groups and 1 carboxyl group, which easily reacts with free radicals to form hydrogen free radicals with antioxidant effects, reduce the activity of hydroxyl groups and superoxide anions, and play an antioxidant role.

[0131] In summary, the appropriate level of CGA added to the functional feed of the present invention is 0.04%, which can improve the growth performance and feed efficiency of Leuciscus walleye, inhibit hepatopancreatic fat deposition through multiple pathways such as enzyme activity and gene regulation, promote fat decomposition to prevent lipid peroxidation in the fish body, activate the NF-κB signaling pathway to reduce the occurrence of inflammatory response, and activate the Keap1-Nrf2 signaling pathway to improve antioxidant capacity.

[0132] The above describes and evaluates the efficacy of some embodiments of the present invention. It should be understood that the present invention is not limited to the above specific implementations. Any technician familiar with the art can use the above disclosed methods and technical contents to make many possible changes and modifications to the technical solutions of the present invention without departing from the scope of the technical solutions of the present invention, or modify them into equivalent embodiments of equivalent changes, which does not affect the essential content of the present invention. Therefore, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solutions of the present invention still falls within the scope of protection of the technical solutions of the present invention.

Claims

1. A functional feed for promoting the hepatopancreas health of Leuciscus walleye, characterized in that: It comprises basic feed and additive chlorogenic acid; the added amount of the chlorogenic acid is 0.02-0.08%.

2. The functional feed according to claim 1, characterized in that The added amount of chlorogenic acid is 0.04%.

3. The functional feed according to claim 1, characterized in that The basic feed comprises the following raw materials in parts by weight: 29.5-30.5 parts of fish meal, 11-12 parts of peeled soybean meal, 9.5-10.5 parts of soybean protein concentrate, 4.5-5.5 parts of corn gluten meal, 17.5-18.5 parts of flour, 2-3 parts of fish oil, 2-3 parts of soybean oil, 0.4-0.6 parts of mineral and vitamin premix, 1.5-2.5 parts of monocalcium phosphate, 0.2-0.4 parts of choline chloride, 0.07-0.09 parts of vitamin C ester, 2.5-3.5 parts of microcrystalline cellulose and 14-15 parts of dextrin.

4. The functional feed according to claim 3, characterized in that The functional feed is made of the following raw materials, calculated by mass percentage: 30% fish meal, 11.53% peeled soybean meal, 10% soybean protein concentrate, 5% corn gluten meal, 18% flour, 2.44% fish oil, 2.44% soybean oil, 0.04% chlorogenic acid, 0.5% mineral and vitamin premix, 2% monocalcium phosphate, 0.3% choline chloride, 0.08% vitamin C ester, 3.03% microcrystalline cellulose and 14.64% dextrin.

5. The functional feed according to claim 3, characterized in that The basic feed has a crude protein content of 36-37%, a crude fat content of 7-8% and a crude ash content of 6-7%.

6. A method for preparing a functional feed according to any one of claims 1 to 5, characterized in that: The following steps are involved: S1. The raw materials of chlorogenic acid and basic feed are crushed and sieved respectively, and then mixed evenly; then put into a screw extruder and extruded into granular feed; S2. The granular feed obtained in step S1 is dried, sealed, and frozen for storage to obtain the functional feed.

7. The preparation method according to claim 6, characterized in that: In step S1, the mesh size of the sieving is 0.2-0.3 mm; and / or the particle diameter of the granular feed is 1.5 mm.

8. The preparation method according to claim 6, characterized in that: In step S2, the drying condition is: drying at 45°C for 24 hours; and / or the freezing storage temperature is -20°C.

9. Use of the functional feed according to any one of claims 1 to 5 in the field of fish farming.

10. The use according to claim 9, characterized in that: The application includes promoting the growth performance, lipid metabolism function, immune function and antioxidant capacity of fish.

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