Novel compound feed for leuciscus waleckii as well as preparation method and application of novel compound feed

Abstract

The invention provides a novel compound feed for leuciscus waleckii, which comprises the following components: 10 to 30 percent of fish meal, 8 to 12 percent of soybean protein concentrate, 15 to 20 percent of flour, 3 to 6 percent of casein, 0.5 to 2 percent of gelatin, 4 to 6 percent of corn protein powder, 2 to 3 percent of soybean oil, 2 to 3 percent of fish oil, 18 to 24 percent of dextrin, 0.3 to 0.6 percent of mineral substance and vitamin premix, 0.2 to 0.6 percent of choline chloride, 1.5 to 2.5 percent of monocalcium phosphate, 0.05 to 0.12 percent of vitamin C ester, 2.5 to 3.5 percent of microcrystalline cellulose and the balance of water. The non-grain novel protein source defatted tenebrio molitor powder is used for partially replacing the fish meal, and the replacement proportion is 0-60%. The invention further provides a preparation method of the novel compound feed for the leuciscus waleckii, and the obtained feed is used for the leuciscus waleckii and can promote the growth performance, the digestive function, the protein metabolism function, the lipid metabolism function, the immune function and the oxidation resistance of the leuciscus waleckii. The invention provides a theoretical reference for developing a novel non-grain protein source, saving fish meal protein, reducing the feed cost and optimizing the artificial compound feed for the leuciscus waleckii.

Description

Technical Field

[0001] The invention belongs to the technical field of aquatic feeds, and in particular relates to a new type of compound feed for Leuciscus walleyeii and a preparation method and application thereof. Background Art

[0002] Fishmeal has become one of the main sources of protein in aquaculture due to its high protein content and balanced nutritional composition. However, in recent years, the shortage of fishmeal, resource shortage and price increase have brought huge cost pressure to the aquaculture industry, seriously restricting the sustainable development of the aquaculture industry. At the same time, population growth and reduced arable land area have led to increasingly serious competition between feed resources and human food. Finding new sustainable sources of protein can reduce the pressure of protein feed deficiency. my country's new non-grain protein sources are rich in resources, diverse in variety, and have the characteristics of sustainable utilization, but their application in aquatic feed has not been fully explored. Therefore, in the context of increasingly scarce global food resources, in order to save the use of fishmeal protein and ensure national food security, it is necessary to rationally develop new non-grain protein sources to alleviate or eliminate the shortage of aquatic feed raw materials.

[0003] Insect protein will become an important protein source to replace fish meal in aquaculture feed in the next 10-20 years. Tenebrio molitor (TM) is an insect of the family Tenebrioidae in the order Coleoptera. It is distributed all over the world and has high protein and lipid content and a reasonable amino acid composition. The European Union approved a Commission proposal for the final regulation and authorization of the use of insect feed in fish feed, in which mealworm is one of the seven insect species. However, since the fat and fiber content in insect meal is higher than that in fish meal, mealworm meal is often defatted to balance processing technology and high protein requirements. In some existing studies, such as largemouth bass (Micropterus salmoides), European sea bass (Decentrarchus labrax L.), Macrobrachium rosenbergii, etc., it has been shown that mealworm meal in feed can partially replace fish meal, and in studies on turbot (Scophthalmus maximus), rainbow trout (Oncorhynchus mykiss), vannamei shrimp (Litopenaeus vannamei) and large yellow croaker (Larimichthys crocea), it has also been shown that defatted mealworm meal can partially replace fish meal and improve the growth performance of aquatic animals. However, there has been no report on the effect of defatted mealworm meal, a new non-grain protein source, replacing fish meal on Leuciscus walleyeii. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a new type of compound feed for Leuciscus walleye and its preparation method and application in view of the deficiencies of the above-mentioned prior art. Defatted yellow mealworm powder is used to replace part of fish meal in the feed, and the effects on the growth performance, digestive function, protein metabolism function, lipid metabolism function, immune function and antioxidant capacity of Leuciscus walleye are evaluated, so as to provide a theoretical reference for developing new non-grain protein sources and saving fish meal protein, which can effectively reduce feed costs and optimize artificial compound feed for Leuciscus walleye.

[0005] In order to solve the above technical problems, the technical solution adopted by the present invention is:

[0006] The invention discloses a novel compound feed for Leuciscus walleye, which comprises the following components: 10-30% of fish meal, 8-12% of soybean protein concentrate, 15-20% of flour, 3-6% of casein, 0.5-2% of gelatin, 4-6% of corn protein powder, 2-3% of soybean oil, 2-3% of fish oil, 18-24% of dextrin, 0.3-0.6% of mineral and vitamin premix, 0.2-0.6% of choline chloride, 1.5-2.5% of calcium dihydrogen phosphate, 0.05-0.12% of vitamin C ester, 2.5-3.5% of microcrystalline cellulose, 0-0.5% of lysine and 0-0.5% of methionine, wherein the fish meal is partially replaced by defatted mealworm powder, a novel non-grain protein source, and the replacement ratio is 0-60%.

[0007] Preferably, the feed comprises the following components: 20-30% fish meal, 9-11% soybean protein concentrate, 17-19% flour, 4-5% casein, 0.5-1.5% gelatin, 4-6% corn gluten meal, 2-3% soybean oil, 2-3% fish oil, 19-21% dextrin, 0.4-0.5% mineral and vitamin premix, 0.2-0.5% choline chloride, 1.8-2.3% calcium dihydrogen phosphate, 0.08-0.1% vitamin C ester, 3.0-3.5% microcrystalline cellulose, 0-0.5% lysine, 0-0.2% methionine, wherein the fish meal is partially replaced by non-grain protein source defatted mealworm powder, and the replacement ratio is 15-45%.

[0008] Preferably, the feed comprises the following components: 20.6% fish meal, 8.66% defatted mealworm powder, 10% soybean protein concentrate, 18% flour, 4.88% casein, 1.22% gelatin, 5.1% corn gluten meal, 2.54% soybean oil, 2.54% fish oil, 20.03% dextrin, 0.5% mineral and vitamin premix, 0.3% choline chloride, 2% monocalcium phosphate, 0.08% vitamin C ester, 3.27% microcrystalline cellulose, 0.2% lysine, and 0.08% methionine.

[0009] Preferably, the mineral and vitamin premix is ​​commercially available.

[0010] Preferably, the crude protein content of the feed is 35-40%, the crude fat content is 7-8%, and the crude ash content is 4-7%.

[0011] The present invention also provides a method for preparing the novel compound feed for Leuciscus walleye, which comprises the following steps: crushing the raw materials and passing them through a 60-mesh sieve, weighing them according to the formula amount, mixing them evenly, using a multifunctional screw extruder to form granular feed with a diameter of 1 to 2 mm, drying them at a temperature of 40 to 50° C. for 20 to 26 hours, sealing them, and then storing them in a refrigerator at -15 to -25° C. for later use.

[0012] The present invention also provides an application of the novel compound feed of Leuciscus variabilis, wherein the feed is used for aquatic feed. The feed is used for artificial compound feed of Leuciscus variabilis, and can promote the growth performance, digestive function, protein metabolism function, lipid metabolism function, immune function and antioxidant capacity of Leuciscus variabilis.

[0013] The present invention has significant technical effects due to the adoption of the above technical solution:

[0014] 1. The present invention provides a new type of compound feed for Leuciscus walleye, and a preparation method and application thereof. The feed adopts defatted mealworm powder, a new type of non-grain protein source, to replace part of fish meal, which has a positive effect on the growth, feed utilization rate, digestive function, protein metabolism function, lipid metabolism function, immune function and antioxidant capacity of Leuciscus walleye, and provides a theoretical basis for replacing fish meal with defatted mealworm powder, a new type of non-grain protein source.

[0015] 2. The novel compound feed of the present invention replaces part of the fish meal with defatted mealworm powder, is suitable for large-scale production, has a wide source, low cost, and is easy to promote. It is helpful to develop a new non-grain protein source, save fish meal protein, alleviate the problem of large fish meal consumption, reduce feed costs, optimize fish feed composition, and promote healthy fish farming.

[0016] The present invention is further described in detail below with reference to the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The effect of defatted mealworm powder replacing fish meal in Example 6 of the present invention on the expression of genes related to the mTOR signaling pathway in the hepatopancreas of Leuciscus walleye;

[0018] Figure 2 The effect of defatted mealworm powder replacing fish meal in Example 6 of the present invention on the expression of genes related to hepatopancreas lipid metabolism in Leuciscus walleye;

[0019] Figure 3 The effect of defatted mealworm powder replacing fish meal in Example 6 of the present invention on the expression of genes related to hepatopancreatic inflammation in Leuciscus walleye;

[0020] Figure 4 The effect of defatted mealworm powder replacing fish meal in Example 6 of the present invention on the activities of antioxidant-related enzymes in the hepatopancreas of Leuciscus walleye;

[0021] Figure 5 The present invention is to use the defatted mealworm meal in Example 6 to replace fish meal, which has an effect on the expression of antioxidant-related genes in the hepatopancreas of Leuciscus walleye. DETAILED DESCRIPTION

[0022] Example 1

[0023] The present embodiment is a novel compound feed for Walleye fish, comprising the following components: 30% fish meal, 10% soybean protein concentrate, 18% flour, 4.88% casein, 1.22% gelatin, 5.1% corn gluten meal, 2.41% soybean oil, 2.41% fish oil, 20.03% dextrin, 0.5% mineral and vitamin premix, 0.3% choline chloride, 2% monocalcium phosphate, 0.08% vitamin C ester, and 3.07% microcrystalline cellulose.

[0024] The mineral and vitamin premixes were purchased from Shenyang Keda Feed Co., Ltd., China, with a mg / kg premix.

[0025] The preparation method is as follows: the raw materials are crushed and passed through a 60-mesh sieve, weighed according to the formula, mixed evenly, and made into 1.5 mm diameter granular feed using a multifunctional screw extruder, dried at 45°C for 24 hours, sealed, and then stored in a -20°C refrigerator for later use.

[0026] Example 2

[0027] This embodiment is a novel compound feed for Walleye fish, comprising the following components: 25.3% fish meal, 4.33% defatted mealworm powder, 10% soybean protein concentrate, 18% flour, 4.88% casein, 1.22% gelatin, 5.1% corn gluten meal, 2.48% soybean oil, 2.48% fish oil, 20.03% dextrin, 0.5% mineral and vitamin premix, 0.3% choline chloride, 2% calcium dihydrogen phosphate, 0.08% vitamin C ester, 3.16% microcrystalline cellulose, 0.1% lysine, and 0.04% methionine. The preparation method is the same as that of Example 1.

[0028] Example 3

[0029] This embodiment is a novel compound feed for Walleye fish, comprising the following components: 20.6% fish meal, 8.66% defatted mealworm powder, 10% soybean protein concentrate, 18% flour, 4.88% casein, 1.22% gelatin, 5.1% corn gluten meal, 2.54% soybean oil, 2.54% fish oil, 20.03% dextrin, 0.5% mineral and vitamin premix, 0.3% choline chloride, 2% calcium dihydrogen phosphate, 0.08% vitamin C ester, 3.27% microcrystalline cellulose, 0.2% lysine, and 0.08% methionine. The preparation method is the same as that of Example 1.

[0030] Example 4

[0031] This embodiment is a novel compound feed for Walleye fish, comprising the following components: 15.88% fish meal, 13% defatted mealworm powder, 10% soybean protein concentrate, 18% flour, 4.88% casein, 1.22% gelatin, 5.1% corn gluten meal, 2.61% soybean oil, 2.61% fish oil, 20.03% dextrin, 0.5% mineral and vitamin premix, 0.3% choline chloride, 2% calcium dihydrogen phosphate, 0.08% vitamin C ester, 3.37% microcrystalline cellulose, 0.3% lysine, and 0.12% methionine. The preparation method is the same as that of Example 1.

[0032] Example 5

[0033] This embodiment is a novel compound feed for Walleye fish, comprising the following components: 11.18% fish meal, 17.33% defatted mealworm powder, 10% soybean protein concentrate, 18% flour, 4.88% casein, 1.22% gelatin, 5.1% corn gluten meal, 2.68% soybean oil, 2.68% fish oil, 20.03% dextrin, 0.5% mineral and vitamin premix, 0.3% choline chloride, 2% calcium dihydrogen phosphate, 0.08% vitamin C ester, 3.46% microcrystalline cellulose, 0.4% lysine, and 0.16% methionine. The preparation method is the same as that of Example 1.

[0034] Example 6

[0035] This example studies the effect of the novel compound feed prepared by the present invention on the growth performance, digestive function, protein metabolism function, lipid metabolism function, immune function and antioxidant capacity of Leuciscus wallichii.

[0036] 1. Preparation of experimental feed

[0037] Fish meal, soybean protein concentrate and defatted mealworm meal were used as the main protein sources, and soybean oil and fish oil were used as fat sources. The defatted mealworm meal replaced fish meal protein in the ratio of (0.0%, 15.0%, 30.0%, 45.0%, 60.0%), and was prepared into 5 kinds of experimental feeds (CK, TM1, TM2, TM3, TM4) with equal nitrogen and equal fat. After crushing, various raw materials were passed through a 60-mesh sieve, weighed according to the feed formula (Table 1), mixed evenly, and made into particles with a particle size of 1.5 mm using a multifunctional screw extruder (YL90L4, Jilin Agricultural University, Changchun), and dried in an oven at 45°C for 24 hours, sealed in a sealed bag, and stored in a -20°C refrigerator for use.

[0038] Table 1 Feed formula (air-dried basis, g / kg)

[0039]

[0040]

[0041] The main nutritional compositions of the five experimental feeds are shown in Table 2.

[0042] Table 2 Main nutritional composition of 5 experimental feeds

[0043]

[0044] 2. Feeding test

[0045] The required Walleye fish were purchased from Yongshun Fishery, Xiangling Village, Meihekou City, and the feeding experiment was conducted in the aquaculture room of the College of Animal Science and Technology, Jilin Agricultural University. Before the formal start of the experiment, the fish were temporarily raised for 15 days, during which they were fed with TM0 group feed to adapt to the breeding environment. Before grouping, they were stopped from feeding for 24 hours, and 450 healthy Walleye fish were randomly divided into 5 groups (3 replicates per group, 30 fish per replicate, 300L plastic tank) for an 8-week feeding experiment. During the feeding period, artificial bait was spread every day, and the fish were fed 3 times (7:00, 12:00 and 17:00) at a feeding rate of 3%-5% of their body weight. 1 / 4-1 / 3 of the water was replaced every day, and the water temperature was maintained at 24-28℃, dissolved oxygen>5mg / L, nitrite<0.05mg / L, ammonia nitrogen<0.30mg / L, and pH between 7.5-8.5.

[0046] After the feeding experiment, the fish were stopped from feeding for 24 hours and anesthetized with 100 mg / L MS-222 (3-aminobenzoic acid ethyl ester, Sigma, USA). The group weight of the fish in 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 and feed utilization. Growth indicators and feed utilization were calculated according to the following formula:

[0047] Mass gain rate (WGR) = (Wt-W0) / W0×100%……………………(1) Specific growth rate (SGR) = (ln Wt - ln W0) / t × 100% (2)

[0048] Feed conversion ratio (FCR) = I t / (Wt-W0)………………(3)

[0049] Feed efficiency (FER) = (Wt - W0) / I t ×100%……………………(4)

[0050] Protein efficiency (PER) = (Wt - W0) / (I t ×P)×100%…………………(5)

[0051] Liver to body ratio (HIS) = W H / Wt×100%……………(6)

[0052] Visceral-to-body ratio (VSI) = W V / Wt×100%…………(7)

[0053] Fullness (CF) = Wt / L 3 ×100………………(8)

[0054] Survival rate (SR) = final number / initial number × 100%……………(9)

[0055] 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).

[0056] 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 and immune indicators. The liver and pancreas were collected, quickly frozen in liquid nitrogen, and stored at -80°C for the determination of digestive enzymes, protein metabolism enzyme activities, lipid metabolism and antioxidant capacity, as well as the determination of gene mRNA expression.

[0057] (1) Enzyme activity assay

[0058] Protease activity was determined by Folin phenol reagent method. Amylase, lipase, alanine aminotransferase (ALT), aspartate aminotransferase (AST), acetyl-CoA carboxylase (ACC), hormone-sensitive lipase (HSL), fatty acid synthase (FAS), triglyceride (TG), total cholesterol (TC), triglyceride lipase (ATGL), carnitine palmitoyltransferase 1 (CPT-1), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), alkaline phosphatase (AKP), acid phosphatase (ACP), lysozyme (LZM), total superoxide dismutase (T-SOD), catalase (CAT) and glutathione peroxidase (GPx) activities, as well as complement 3 (C3), complement 4 (C4), immunoglobulin M (IgM), malondialdehyde (MDA), reduced glutathione (GSH), and total antioxidant capacity (T-AOC) were all determined by kits.

[0059] (2) Real-time quantitative PCR analysis

[0060] 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 3 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.

[0061] Table 3 Gene primer sequences

[0062]

[0063]

[0064] The obtained data were analyzed using SPSS (27.0). First, the normality and homogeneity of variance of the data were tested. Secondly, the significance was analyzed by one-way variance analysis (ANOVA), and orthogonal polynomial comparison was used to determine whether there was a significant linear relationship (Linear) and quadratic relationship (Quadratic) between the level of biofloc meal replacing fish meal and related indicators. If the difference in variance analysis was significant, Tukey's multiple comparison was performed. The difference was significant at P < 0.05, and the data were expressed as (mean ± standard error).

[0065] 3. Effect of defatted mealworm meal replacing fish meal on the growth performance of Leuciscus walleye

[0066] As shown in Table 4, with the increase of defatted mealworm meal replacing fish meal, the FW, WGR, SGR, PER and FER of Leuciscus walleye showed a significant quadratic relationship of first increasing and then decreasing (P<0.05). Compared with the TM0 group, the FW, WGR and SGR of the TM1, TM2 and TM3 groups increased significantly (P<0.05), while there was no significant difference in the TM4 group (P>0.05); the PER and FER of the TM2 and TM3 groups increased significantly (P<0.05), while there was no significant difference in the TM1 and TM4 groups (P>0.05). However, there was no significant difference in VSI, HSI, CF and SR among the groups (P>0.05).

[0067] Table 4 Effects on growth and feed utilization of Leuciscus vachelli

[0068]

[0069] Note: Data in the same row with different letters indicate significant differences (P<0.05). (Same as the following table).

[0070] The results of this experiment show that when the defatted mealworm meal replaces fish meal at a level of 30%, the body weight, mass gain rate, protein efficiency, feed efficiency and specific growth rate of Walleye fish are significantly increased, indicating that the appropriate level of defatted mealworm meal replacing fish meal can promote the growth performance and feed utilization of Walleye fish.

[0071] 4. Effects of defatted mealworm meal replacing fish meal on digestive function and protein metabolism of Leuciscus walleye

[0072] As shown in Table 5, the level of defatted mealworm meal replacing fish meal had significant linear and quadratic effects on the protease activity in the foregut of Leuciscus walleye (P<0.05), and significant linear effects on the hepatopancreas and hindgut (P<0.05). Compared with the TM0 group, the protease activity in the hepatopancreas, foregut and hindgut of the TM4 group decreased significantly (P<0.05), while there was no significant difference in the TM1, TM2 and TM3 groups (P>0.05); the protease activity in the midgut was not significantly different from that in the TM0 group (P>0.05).

[0073] Table 5 Effects on protease of Leuciscus wallichiana

[0074]

[0075] As shown in Table 6, there was no significant difference in the lipase activity of the hepatopancreas, foregut, midgut and hindgut of Leuciscus walleye treated with defatted mealworm meal instead of fish meal compared with the TM0 group (P>0.05).

[0076] Table 6 Effects on lipase of Leuciscus walleye

[0077]

[0078]

[0079] As shown in Table 7, there was no significant difference in the amylase activity of the hepatopancreas, foregut, midgut and hindgut of Leuciscus walleye treated with defatted mealworm meal instead of fish meal compared with the TM0 group (P>0.05).

[0080] Table 7 Effects on amylase of Leuciscus walleye

[0081]

[0082] As shown in Table 8, the level of defatted mealworm meal replacing fish meal had a significant quadratic effect on the hepatopancreas ALT and AST enzyme activities of Leuciscus walleye, showing a trend of first increasing and then decreasing (P<0.05); while the serum ALT and AST enzyme activities showed a quadratic trend of first decreasing and then increasing (P<0.05). Compared with the TM0 group, the hepatopancreas ALT and AST enzyme activities of the TM2 and TM3 groups were significantly increased (P<0.05), on the contrary, the serum AST enzyme activities of the TM2 and TM3 groups were significantly decreased (P<0.05); the serum ALT activity of the TM2 and TM4 groups was significantly decreased (P<0.05).

[0083] Table 8 Effects on the activities of enzymes related to protein metabolism in Leuciscus variabilis

[0084]

[0085]

[0086] Depend on Figure 1It can be seen that the level of defatted mealworm meal replacing fish meal had a significant quadratic and linear effect on the mRNA levels of S6K and AKT in the hepatopancreas of Leucissima walleye, showing a trend of first increasing and then decreasing (P<0.05), and had a significant quadratic effect on the mRNA expression levels of mTOR, 4E-BP, PI3K and IGF-1 (P<0.05). The mRNA levels of S6K and AKT in the hepatopancreas of Leucissima walleye in TM2 and TM3 groups were significantly higher than those in the TM0 group (P<0.05), while the mRNA level of 4E-BP was significantly lower than that in the TM0 group (P<0.05). Compared with the TM0 group, the mRNA levels of mTOR and PI3K in the hepatopancreas of the TM2 group increased significantly (P<0.05); the mRNA level of IGF-1 in the TM1, TM2 and TM3 groups increased significantly (P<0.05).

[0087] In this study, when the defatted mealworm meal replaced fish meal at a level of 60% in the TM4 group, the protease activity in the hepatopancreas, foregut, midgut and hindgut of Walleye fish was significantly reduced, affecting the fish's ability to digest and absorb nutrients in the feed. Substituting defatted mealworm meal for 30% of fish meal reduced the activity of AST and ALT in serum, while increasing the activity of AST and ALT in the hepatopancreas, indicating that defatted mealworm meal replacing fish meal can not only maintain the health of the hepatopancreas of Walleye fish, but also improve protein utilization efficiency. The results show that replacing 30% of fish meal with defatted mealworm meal can improve protein metabolic enzyme activity. In addition, replacing 30% fish meal with defatted mealworm meal significantly upregulated the mTOR, S6K, PI3K, AKT and IGF-1 mRNA in the hepatopancreas of Yarrowia walleye, and downregulated the 4E-BP mRNA level, revealing that defatted mealworm meal promoted protein synthesis by activating the mTOR signaling pathway, that is, replacing 30% fish meal with defatted mealworm meal activated the PI3K-AKT-mTOR signaling pathway, promoted the synthesis of hepatopancreas protein, and thus promoted the growth and feed utilization of Yarrowia walleye.

[0088] 5. Effects of defatted mealworm meal replacing fish meal on lipid metabolism function and related gene expression of Yarrowia walleyeii

[0089] As shown in Table 9, with the increase of defatted mealworm meal replacing fish meal, serum FAS, TG and TC activities showed significant linear and quadratic changes (P<0.05), and HSL and CPT-1 activities showed significant linear increasing changes (P<0.05). Compared with the TM0 group, the HSL and CPT-1 activities in the serum TM3 and TM4 groups increased significantly (P<0.05), while the TG and TC activities decreased significantly (P<0.05), while there were no significant differences in the HSL, CPT-1, TG and TC activities in the TM1 and TM2 groups (P>0.05); the FAS enzyme activity in the TM4 group decreased significantly (P<0.05), and there were no significant differences among the other groups (P>0.05). Different levels of defatted mealworm meal replacing fish meal had no significant effect on the ACC activity of Walleye (P>0.05).

[0090] Table 9 Effects of Leuciscus variabilis on serum lipid metabolism-related enzyme activities

[0091]

[0092] As shown in Table 10, the replacement of fish meal with defatted mealworm meal at different levels significantly changed the activities of ACC and TG in the hepatopancreas of Walleye (P<0.05), and the activities of FAS, ATGL, LDL-C and HDL-C showed significant linear and quadratic changes (P<0.05). The activities of ACC, FAS, TG and LDL-C in the hepatopancreas of Walleye TM3 and TM4 groups were significantly lower than those in the TM0 group (P<0.05). On the contrary, the activity of HDL-C was significantly higher than that in the TM0 group (P<0.05). The activity of ATGL in the TM4 group was significantly higher than that in the TM0 group (P<0.05), and there was no significant difference among the other groups (P>0.05).

[0093] Table 10 Effects of Leuciscus variabilis on the activities of enzymes related to hepatopancreas lipid metabolism

[0094]

[0095] like Figure 2 It can be seen that the replacement of fish meal with defatted mealworm meal had significant linear and quadratic changes in the mRNA levels of FAS, PPAR-β and PPAR-γ in the hepatopancreas of Leucissima walleye (P<0.05), and the mRNA level of ACC showed a significant linear decrease (P<0.05). Compared with the TM0 group, the mRNA levels of ACC, FAS and PPAR-γ in the hepatopancreas of the TM3 and TM4 groups were significantly decreased (P<0.05), while the mRNA level of PPAR-β was significantly increased (P<0.05), and there was no significant difference among the other groups (P>0.05).

[0096] This study found that defatted mealworm meal replacing 45% and 60% of fish meal led to a significant decrease in serum TG and TC enzyme activities, indicating that defatted mealworm meal replacing 45%-60% of fish meal in the feed can reduce the serum fat level of Walleye. After defatted mealworm meal replaced 45% and 60% of fish meal in the feed, the activities of ACC, FAS and LDL-C in the hepatopancreas of Walleye significantly decreased, while the activities of ATGL and HDL-C significantly increased. At the gene level, it also showed that defatted mealworm meal replacing fish meal can regulate lipid metabolism by promoting lipolysis genes (including PPAR-β) and inhibiting lipid synthesis genes (including ACC, PPAR-γ and FAS). These results reveal that defatted mealworm meal can promote lipid decomposition and inhibit lipid synthesis in Walleye, reducing fat deposition in the hepatopancreas.

[0097] 6. Effects of defatted mealworm meal replacing fish meal on immune function and expression of inflammation-related genes in Leuciscus walleye

[0098] As shown in Table 11, with the increase of defatted mealworm meal replacing fish meal, the serum IgM, C3 and C4 contents of Leuciscus walleye showed significant linear and quadratic changes (P<0.05), AKP activity showed significant linear changes (P<0.05), and LZM enzyme activity showed significant quadratic changes (P<0.05). Compared with the TM0 group, the IgM content of the TM1 and TM2 groups increased significantly (P<0.05), while there was no significant difference between the TM3 and TM4 groups (P>0.05); the LZM and C3 enzyme activities of the TM2 group increased significantly (P<0.05), the C3 enzyme activity of the TM4 group decreased significantly (P<0.05), and the LZM enzyme activity decreased but the difference was not significant (P>0.05); the C4 and AKP activities of the TM4 group decreased significantly (P<0.05), and there was no significant difference between the other groups (P>0.05). Substituting different levels of defatted mealworm meal for fish meal had no significant effect on the ACP activity of Leuciscus walleye (P>0.05).

[0099] Table 11 Effects on serum immune indexes of Leuciscus walleye

[0100]

[0101] Depend on Figure 3It can be seen that the level of defatted mealworm meal replacing fish meal had significant linear and quadratic effects on the mRNA levels of NF-κBp65, Myd88, TGF-β and TNF-α in the hepatopancreas of Leucissima walleye (P<0.05). The mRNA levels of NF-κBp65 and TNF-α in the hepatopancreas of the TM2 group were significantly decreased compared with the TM0 group (P<0.05), and significantly increased in the TM4 group (P<0.05), while there was no significant difference between the TM1 and TM3 groups and the TM0 group (P>0.05). The mRNA level of Myd88 in the hepatopancreas of the TM4 group was significantly increased compared with the TM0 group (P<0.05), while there was no significant difference between the TM1, TM2 and TM3 groups and the TM0 group (P>0.05). The hepatopancreas TGF-β mRNA level in TM2 group was significantly upregulated compared with TM0 group (P<0.05), and was significantly downregulated in TM4 group compared with TM0 group (P<0.05), while there was no significant difference between TM1 and TM3 groups and TM0 group (P>0.05).

[0102] The results showed that after replacing fish meal with 30% defatted mealworm meal, the serum LZM, IgM and C3 enzyme activities of Yarrowia walleye were significantly increased, and the NF-κBp65 and TNF-α mRNA levels in the hepatopancreas of Yarrowia walleye were downregulated, and the TGF-β mRNA level was upregulated; but when the defatted mealworm meal replaced fish meal at a ratio of 60%, the hepatopancreas MyD88, NF-κBp65, and TNF-α mRNA levels were upregulated, and the TGF-β mRNA level was downregulated, indicating that defatted mealworm meal replacing fish meal at an appropriate level effectively inhibited the activation of the NF-κB signaling pathway and improved the immune function of Yarrowia walleye, while high-level defatted mealworm meal replacing fish meal promoted the inflammatory response of the fish by activating the NF-κB signaling pathway.

[0103] 7. Effects of defatted mealworm meal replacing fish meal on antioxidant capacity and related gene expression of Leuciscus walleye

[0104] Depend on Figure 4It can be seen that with the increase of defatted mealworm meal replacing fish meal, the MDA content and T-AOC and CAT levels in the hepatopancreas of Leucissima walleye showed significant quadratic and linear relationships (P<0.05), and the GPX and T-SOD activities had significant quadratic effects (P<0.05). The GPX and T-AOC activities of TM1 and TM2 groups were significantly increased compared with TM0 group (P<0.05), and there was no significant difference between TM3 and TM4 groups compared with TM0 group (P>0.05). Compared with TM0 group, the MDA content of TM1 group decreased significantly (P<0.05), and that of TM4 group increased significantly (P<0.05); the T-SOD activity of TM2 group increased significantly (P<0.05), and there was no significant difference between TM1, TM3, and TM4 groups compared with TM0 group (P>0.05); the CAT activity of TM2 group increased significantly (P<0.05), and that of TM4 group decreased significantly (P<0.05). There was no significant difference in hepatopancreas GSH level compared with the TM0 group (P>0.05).

[0105] Depend on Figure 5 It can be seen that with the increase of defatted mealworm meal replacing fish meal, the mRNA levels of Keap1, HO-1, Nrf2 and SOD in the hepatopancreas of Leucissima walleye showed significant linear and quadratic changes (P<0.05), the mRNA level of GPX-4 showed a significant quadratic change (P<0.05), and the mRNA level of CAT showed a significant linear effect (P<0.05). The mRNA levels of CAT, HO-1, Nrf2 and SOD in the hepatopancreas of TM2, TM3 and TM4 groups were significantly increased compared with those of TM0 group (P<0.05), while there was no significant difference between TM1 group and TM0 group (P>0.05). The mRNA level of GPX-4 in TM1, TM2 and TM3 groups was significantly higher than that in TM0 group (P<0.05), while there was no significant effect between TM4 group and TM0 group (P>0.05). The hepatopancreas Keap1 mRNA levels in the TM2, TM3, and TM4 groups were significantly decreased compared with those in the TM0 group (P<0.05), while there was no significant difference between the TM1 group and the TM0 group (P>0.05).

[0106] The results showed that defatted mealworm meal replacing 30% fish meal increased the activities of T-AOC, GPX, GSH, CAT and T-SOD in the hepatopancreas of Walleye, and reduced the MDA content, revealing that defatted mealworm meal partially replaced fish meal to enhance the antioxidant capacity. The antioxidant effect of defatted mealworm meal involved the Nrf2 pathway. When replacing 15%-30% fish meal, the levels of Nrf2, GPX-4, HO-1, SOD and CAT mRNA in the hepatopancreas were increased, and the level of Keap1 mRNA was reduced, revealing that defatted mealworm meal can activate the Keap1 / Nrf2 signaling pathway, increase the expression levels of downstream genes such as SOD and CAT mRNA by upregulating the expression of Nrf2 mRNA, and thus enhance the antioxidant capacity of Walleye.

[0107] The above is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent change made to the above embodiment according to the technical essence of the invention still falls within the protection scope of the technical solution of the present invention.

Claims

1. A novel compound feed for Leuciscus walleye, characterized in that: The feed comprises the following components: 10-30% fish meal, 8-12% soybean protein concentrate, 15-20% flour, 3-6% casein, 0.5-2% gelatin, 4-6% corn protein powder, 2-3% soybean oil, 2-3% fish oil, 18-24% dextrin, 0.3-0.6% mineral and vitamin premix, 0.2-0.6% choline chloride, 1.5-2.5% calcium dihydrogen phosphate, 0.05-0.12% vitamin C ester, 2.5-3.5% microcrystalline cellulose, 0-0.5% lysine, 0-0.5% methionine, wherein the fish meal is partially replaced by defatted mealworm powder, a new type of non-grain protein source, and the replacement ratio is 0-60%.

2. The feed according to claim 1, characterized in that The feed comprises the following components: 20-30% of fish meal, 9-11% of soybean protein concentrate, 17-19% of flour, 4-5% of casein, 0.5-1.5% of gelatin, 4-6% of corn protein powder, 2-3% of soybean oil, 2-3% of fish oil, 19-21% of dextrin, 0.4-0.5% of mineral and vitamin premix, 0.2-0.5% of choline chloride, 1.8-2.3% of calcium dihydrogen phosphate, 0.08-0.1% of vitamin C ester, 3.0-3.5% of microcrystalline cellulose, 0-0.5% of lysine, and 0-0.2% of methionine, wherein the fish meal is partially replaced by non-grain protein source defatted mealworm powder, and the replacement ratio is 15-45%.

3. The feed according to claim 2, characterized in that 30% of fish meal is replaced by defatted mealworm powder, a non-grain protein source. The feed comprises the following components: 20.6% of fish meal, 8.66% of defatted mealworm powder, 10% of soybean protein concentrate, 18% of flour, 4.88% of casein, 1.22% of gelatin, 5.1% of corn protein powder, 2.54% of soybean oil, 2.54% of fish oil, 20.03% of dextrin, 0.5% of mineral and vitamin premix, 0.3% of choline chloride, 2% of calcium dihydrogen phosphate, 0.08% of vitamin C ester, 3.27% of microcrystalline cellulose, 0.2% of lysine and 0.08% of methionine.

4. The feed according to any one of claims 1 to 3, characterized in that The mineral and vitamin premixes are commercially available.

5. The feed according to any one of claims 1 to 4, characterized in that The crude protein content of the feed is 35-40%, the crude fat content is 7-8% and the crude ash content is 4-7%.

6. A method for preparing the novel compound feed for Leuciscus walleye as claimed in any one of claims 1 to 5, characterized in that: The raw materials are crushed and passed through a 60-mesh sieve, weighed according to the formula, mixed evenly, and made into granular feed with a diameter of 1 to 2 mm using a multifunctional screw extruder. After drying at a temperature of 40 to 50°C for 20 to 26 hours, the granules are sealed and then stored in a refrigerator at -15 to -25°C for later use.

7. Use of the novel compound feed for Leuciscus walleye as claimed in any one of claims 1 to 5, characterized in that: The feed is used for aquatic feed.

8. The use according to claim 7, characterized in that: The feed is used in artificial feed of Leuciscus vachelli and can promote the growth performance, digestive function, protein metabolism function, lipid metabolism function, immune function and antioxidant capacity of Leuciscus vachelli.

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