A kind of Heilongjiang grayling juvenile fish feed and Heilongjiang grayling juvenile fish breeding method

Abstract

The present invention relates to a Heilongjiang grayling fry feed and a method for raising Heilongjiang grayling fry, and belongs to the technical field of feeding feed. To solve the problem that existing fry feeds cannot meet the growth and development needs of Heilongjiang grayling fry, the present invention provides a Heilongjiang grayling fry feed, wherein the total fat content in the feed is 150-200 g / kg and the total protein content is 475-477 g / kg. The Heilongjiang grayling fry feed provided by the present invention has a balanced and reasonable ratio of fat and protein, which can prevent the protein in the feed from being used as an energy source and avoid the harmful effects of excessive fat deposition in the fry. When the feed of the present invention is used for raising Heilongjiang grayling fry, it is beneficial to the growth and health of the Heilongjiang grayling fry and improves the survival rate of the Heilongjiang grayling fry.

Description

Technical Field

[0001] The invention belongs to the technical field of feeding, and in particular relates to a feed for Heilongjiang grayling juveniles and a method for raising Heilongjiang grayling juveniles. Background Art

[0002] Heilongjiang grayling (Thymallus grubii), a small, carnivorous, cold-water fish belonging to the family Grunniidae, is a Salmoniformes order. Its tender, delicious meat boasts high nutritional and edible value. Its meat is low in cholesterol and rich in amino acids, unsaturated fatty acids, minerals, and vitamins, making it a valuable cold-water fish. However, habitat loss and human activities are threatening wild populations of Heilongjiang grayling. In recent years, large numbers of juvenile Heilongjiang grayling have been released into the Heilongjiang River basin annually to restore the dwindling population and maintain biodiversity.

[0003] Carnivorous fish cannot efficiently utilize carbohydrates, and fat is considered the primary energy source in the diet that affects the growth, metabolism, and health of cold-water fish. Insufficient fat in the diet leads to protein consumption as energy. When the amount of essential fatty acids and fat-soluble vitamins provided by fat fails to meet the growth needs of juvenile fish, it can reduce fish growth performance and cause metabolic disorders and disease. However, excessive fat intake in the diet can lead to severe lipid accumulation in tissues, inflammatory responses, cell apoptosis, and intestinal flora disturbances, inhibiting fish growth and feed utilization, and reducing muscle quality.

[0004] At present, there is no special formula feed for Heilongjiang grayling juveniles. Therefore, based on a full understanding of the nutritional needs of Heilongjiang grayling juveniles, research on feed and feeding methods suitable for the growth and development of Heilongjiang grayling juveniles is of great significance to the protection, development and utilization of Heilongjiang grayling. Summary of the Invention

[0005] In order to solve the problem that existing juvenile fish feed cannot meet the growth and development requirements of Heilongjiang grayling juveniles, the present invention provides a Heilongjiang grayling juvenile fish feed and a Heilongjiang grayling juvenile fish breeding method.

[0006] The technical solution of the present invention:

[0007] A feed for Heilongjiang grayling juveniles, comprising a total fat content of 150-200 g / kg and a total protein content of 475-477 g / kg. Per 1000 parts by weight, the feed for Heilongjiang grayling juveniles comprises the following components in parts by weight: 400 parts of fish meal, 40 parts of corn protein, 150 parts of soybean protein concentrate, 85 parts of wheat gluten, 109.8 parts of flour, 120-150 parts of fish oil, 10 parts of a vitamin and mineral premix, 30-60 parts of cellulose, 10 parts of methyl cellulose, 10 parts of sodium alginate, 5 parts of choline, and 0.2 parts of 2,6-di-tert-butyl-p-cresol.

[0008] Furthermore, the total fat content of the feed is 185.3 g / kg, the total protein content is 476.5 g / kg, and every 1,000 parts by weight of the Heilongjiang grayling juvenile feed contains the following components in parts by weight: 400 parts of fish meal, 40 parts of corn protein, 150 parts of soybean protein concentrate, 85 parts of gluten, 109.8 parts of flour, 150 parts of fish oil, 10 parts of vitamin and mineral premix, 30 parts of cellulose, 10 parts of methyl cellulose, 10 parts of sodium alginate, 5 parts of choline, and 0.2 parts of 2,6-di-tert-butyl-p-cresol.

[0009] Furthermore, each kg of the vitamin and mineral premix contains VA 750000 IU, VD3 200000 IU, VE 6000 mg, VK3 2000 mg, VB1 1200 mg, VB2 1200 mg, VB6 1200 mg, VB 12 8 mg, VC 21000 mg, d-calcium pantothenate 2000 mg, niacinamide 9000 mg, folic acid 370 mg, biotin 15 mg, inositol 10000 mg, MgSO4 6000 mg, ZnSO4 4000 mg, MnSO4 2500 mg, CuSO4 2500 mg, FeSO4 2500 mg, CoSO4 160 mg, Ca(IO3)2 200 mg and Na2SeO3 40 mg.

[0010] Furthermore, the preparation method of the Heilongjiang grayling juvenile feed is as follows: fish meal, corn protein, soy protein concentrate, gluten, flour, vitamin and mineral premix, cellulose, methyl cellulose, sodium alginate, choline and 2,6-di-tert-butyl-p-cresol are all passed through a 60-mesh sieve, weighed and mixed evenly, fish oil and water are added, mixed evenly again to make a granular feed, dried to a moisture content of less than 10%, and stored under low-temperature drying conditions for future use.

[0011] A method for raising Heilongjiang grayling juveniles comprises the following steps: in an indoor recirculating aquaculture system, maintaining a water temperature of 12.5-13°C, maintaining dissolved oxygen at no less than 8 mg / L, carrying a photoperiod of 12 hours light / 12 hours dark, changing water by 20% daily, and feeding Heilongjiang grayling juveniles with a feed twice daily until the fish appear full.

[0012] Furthermore, the total fat content of the Heilongjiang grayling juvenile feed is 150-200 g / kg, the total protein content is 475-477 g / kg, and every 1000 parts by weight of the Heilongjiang grayling juvenile feed contains the following components in parts by weight: 400 parts of fish meal, 40 parts of corn protein, 150 parts of soybean protein concentrate, 85 parts of gluten, 109.8 parts of flour, 120-150 parts of fish oil, 10 parts of vitamin and mineral premix, 30-60 parts of cellulose, 10 parts of methyl cellulose, 10 parts of sodium alginate, 5 parts of choline, and 0.2 parts of 2,6-di-tert-butyl-p-cresol.

[0013] Furthermore, the total fat content of the Heilongjiang grayling juvenile feed is 185.3 g / kg, the total protein content is 476.5 g / kg, and every 1000 parts by weight of the Heilongjiang grayling juvenile feed contains the following components in parts by weight: 400 parts of fish meal, 40 parts of corn protein, 150 parts of soybean protein concentrate, 85 parts of gluten, 109.8 parts of flour, 150 parts of fish oil, 10 parts of vitamin and mineral premix, 30 parts of cellulose, 10 parts of methyl cellulose, 10 parts of sodium alginate, 5 parts of choline, and 0.2 parts of 2,6-di-tert-butyl-p-cresol.

[0014] Furthermore, each kg of the vitamin and mineral premix contains VA 750000 IU, VD3 200000 IU, VE 6000 mg, VK3 2000 mg, VB1 1200 mg, VB2 1200 mg, VB6 1200 mg, VB 12 8 mg, VC 21000 mg, d-calcium pantothenate 2000 mg, niacinamide 9000 mg, folic acid 370 mg, biotin 15 mg, inositol 10000 mg, MgSO4 6000 mg, ZnSO4 4000 mg, MnSO4 2500 mg, CuSO4 2500 mg, FeSO4 2500 mg, CoSO4 160 mg, Ca(IO3)2 200 mg and Na2SeO3 40 mg.

[0015] Furthermore, the feeding time is 8:30 and 16:00 every day.

[0016] Beneficial effects of the present invention:

[0017] The fat and protein ratio in the Heilongjiang grayling juvenile feed provided by the present invention is balanced and reasonable, which can prevent the protein in the feed from being used as an energy source and avoid the harmful effects of excessive fat deposition in the juvenile fish. The feed of the present invention can improve the specific growth rate and weight gain of Heilongjiang grayling juveniles, improve protein efficiency and reduce feed conversion, increase the activities of trypsin, lipase, and catalase in the intestines of Heilongjiang grayling juveniles and superoxide dismutase, catalase, alkaline phosphatase, and acid phosphatase in the liver, reduce the level of NF-κB mRNA in the liver, avoid obvious vacuolation of hepatocytes caused by excessive lipid intake, and do not cause a decrease in the abundance of beneficial bacteria and an increase in the abundance of harmful bacteria in the intestine. When the feed of the present invention is used for the rearing of Heilongjiang grayling juveniles, it is beneficial to the growth and health of the Heilongjiang grayling juveniles and improves the survival rate of the Heilongjiang grayling juveniles. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 These are histological staining photos of the livers of Heilongjiang grayling juveniles fed with each group of feeds. Figure a is for Comparative Example 1, Figure b is for Comparative Example 2, Figure c is for Example 2, Figure d is for Example 1, and Figure e is for Comparative Example 3. N represents hepatocyte nuclei, and V represents lipid vesicles.

[0019] Figure 2 This is a comparison of the expression of genes related to lipid metabolism in the liver of Heilongjiang grayling juveniles fed with different groups of feed;

[0020] Figure 3 The figure is a comparison of the expression of immune-related genes in the liver of Heilongjiang grayling juveniles fed with different groups of feeds;

[0021] Figure 4 The following is a comparison of the expression of immune-related genes in the intestine of Heilongjiang grayling juveniles fed with different groups of feed;

[0022] Figure 5 This is a comparison chart of the Sobs index of the rarefaction curve of the intestinal microbial diversity of Heilongjiang grayling juveniles fed with different groups of feed;

[0023] Figure 6 This is a comparison chart of the Shannon index of the rarefaction curve of the intestinal microbial diversity of Heilongjiang grayling juveniles fed with different groups of feed;

[0024] Figure 7 The results of principal coordinate analysis of the intestinal microbial community at the genus level based on the binary Jaccard index of the intestinal microorganisms of Heilongjiang grayling juveniles fed with each group of feeds;

[0025] Figure 8 The principal coordinate analysis results of the intestinal microbial community at the genus level based on the weighted Unifrac index of the intestinal microorganisms of Heilongjiang grayling juveniles fed with each group of feed;

[0026] Figure 9 The results of the Venn diagram analysis of shared and unique OTUs among intestinal microorganisms of Heilongjiang grayling juveniles fed with each group of feeds at the genus level;

[0027] Figure 10 This is a comparison of the relative abundance of intestinal microorganisms at the phylum level in Heilongjiang grayling juveniles fed with different feed groups;

[0028] Figure 11 This is a comparison of the relative abundance of intestinal microorganisms at the genus level in Heilongjiang grayling juveniles fed with different feed groups;

[0029] Figure 12 This is the LEfSe analysis result of the difference in the abundance of intestinal microbial taxa in Heilongjiang grayling juveniles fed with different diet groups;

[0030] Figure 13 This is a bar graph of the Kruskal-Wallis H test for the specific microbial composition at the genus level in the intestinal microbiome of Heilongjiang grayling juveniles fed with each diet group;

[0031] Figure 14 This is a comparison chart of the relative abundance differences of genera and species among groups of intestinal microorganisms of Heilongjiang grayling juveniles fed with different groups of feed using the Kruskal-Wallis H test. DETAILED DESCRIPTION

[0032] The technical solution of the present invention is further described below with reference to the embodiments, but is not limited thereto. Any modification or equivalent replacement of the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention shall be included in the scope of protection of the present invention. The process equipment or devices not specifically noted in the following examples are all conventional equipment or devices in the art. Unless otherwise specified, the raw materials used in the examples of the present invention can be obtained commercially; unless otherwise specified, the technical means used in the examples of the present invention are all conventional means well known to those skilled in the art.

[0033] Example 1

[0034] This embodiment provides a feed for Heilongjiang grayling juveniles, wherein the feed has a total fat content of 185.3 g / kg and a total protein content of 476.5 g / kg. Per 1000 parts by weight, the feed for Heilongjiang grayling juveniles contains the following components in parts by weight: 400 parts of fish meal, 40 parts of corn gluten, 150 parts of soy protein concentrate, 85 parts of wheat gluten, 109.8 parts of flour, 150 parts of fish oil, 10 parts of a vitamin and mineral premix, 30 parts of cellulose, 10 parts of methyl cellulose, 10 parts of sodium alginate, 5 parts of choline, and 0.2 parts of 2,6-di-tert-butyl-p-cresol.

[0035] Each kg of the vitamin and mineral premix used in this example contains VA 750000 IU, VD3 200000 IU, VE 6000 mg, VK3 2000 mg, VB1 1200 mg, VB2 1200 mg, VB6 1200 mg, VB 12 8 mg, VC21000 mg, d-calcium pantothenate 2000 mg, niacinamide 9000 mg, folic acid 370 mg, biotin 15 mg, inositol 10000 mg, MgSO4 6000 mg, ZnSO4 4000 mg, MnSO4 2500 mg, CuSO4 2500 mg, FeSO4 2500 mg, CoSO4 160 mg, Ca(IO3)2 200 mg and Na2SeO3 40 mg.

[0036] The fish meal used in this example was purchased from TASA Fish Product Co., Ltd., Peru; fish oil was purchased from Foshan Damao Feed Co., Ltd.; cellulose content >99.0% was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; methylcellulose was purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.; choline content 50% was purchased from Jujia Biotechnology Co., Ltd.; 2,6-di-tert-butyl-p-cresol content >99.0% was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0037] In this embodiment, the preparation method of the Heilongjiang grayling feed is as follows: fish meal, corn protein, soy protein concentrate, gluten, flour, vitamin and mineral premix, cellulose, methylcellulose, sodium alginate, choline and 2,6-di-tert-butyl-p-cresol are all passed through a 60-mesh sieve, weighed and mixed evenly, fish oil and water are added, mixed evenly again, and pelletized into granules with a particle size of 1 mm using a granulator (Tongli Grain Machine, HKJ-218). The pellets are dried in a 60°C forced air drying oven to a moisture content of less than 10%, cooled to room temperature, packed into sealed bags, and stored at -20°C for later use.

[0038] Example 2

[0039] This embodiment provides a feed for Heilongjiang grayling juveniles, wherein the feed has a total fat content of 157.3 g / kg and a total protein content of 475.1 g / kg. Per 1000 parts by weight, the feed for Heilongjiang grayling juveniles contains the following components in parts by weight: 400 parts of fish meal, 40 parts of corn gluten, 150 parts of soy protein concentrate, 85 parts of wheat gluten, 109.8 parts of flour, 120 parts of fish oil, 10 parts of a vitamin and mineral premix, 60 parts of cellulose, 10 parts of methyl cellulose, 10 parts of sodium alginate, 5 parts of choline, and 0.2 parts of 2,6-di-tert-butyl-p-cresol.

[0040] Each kg of the vitamin and mineral premix used in this example contains VA 750000 IU, VD3 200000 IU, VE 6000 mg, VK3 2000 mg, VB1 1200 mg, VB2 1200 mg, VB6 1200 mg, VB 12 8 mg, VC21000 mg, d-calcium pantothenate 2000 mg, niacinamide 9000 mg, folic acid 370 mg, biotin 15 mg, inositol 10000 mg, MgSO4 6000 mg, ZnSO4 4000 mg, MnSO4 2500 mg, CuSO4 2500 mg, FeSO4 2500 mg, CoSO4 160 mg, Ca(IO3)2 200 mg and Na2SeO3 40 mg.

[0041] The fish meal used in this example was purchased from TASA Fish Product Co., Ltd., Peru; fish oil was purchased from Foshan Damao Feed Co., Ltd.; cellulose content >99.0% was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; methylcellulose was purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.; choline content 50% was purchased from Jujia Biotechnology Co., Ltd.; 2,6-di-tert-butyl-p-cresol content >99.0% was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0042] In this embodiment, the preparation method of the Heilongjiang grayling feed is as follows: fish meal, corn protein, soy protein concentrate, gluten, flour, vitamin and mineral premix, cellulose, methylcellulose, sodium alginate, choline and 2,6-di-tert-butyl-p-cresol are all passed through a 60-mesh sieve, weighed and mixed evenly, fish oil and water are added, mixed evenly again, and pelletized into granules with a particle size of 1 mm using a granulator (Tongli Grain Machine, HKJ-218). The pellets are dried in a 60°C forced air drying oven to a moisture content of less than 10%, cooled to room temperature, packed into sealed bags, and stored at -20°C for later use.

[0043] Comparative Example 1

[0044] This comparative example provides a feed, wherein the total fat content of the feed is 103.5 g / kg, the total protein content is 476.6 g / kg, and every 1000 parts by weight of the feed contains the following components in parts by weight: 400 parts of fish meal, 40 parts of corn protein, 150 parts of soybean protein concentrate, 85 parts of gluten, 109.8 parts of flour, 60 parts of fish oil, 10 parts of vitamin and mineral premix, 120 parts of cellulose, 10 parts of methyl cellulose, 10 parts of sodium alginate, 5 parts of choline, and 0.2 parts of 2,6-di-tert-butyl-p-cresol.

[0045] Each kg of the vitamin and mineral premix used in this comparative example contains VA 750000 IU, VD3 200000 IU, VE 6000 mg, VK3 2000 mg, VB1 1200 mg, VB2 1200 mg, VB6 1200 mg, VB 12 8 mg, VC21000 mg, d-calcium pantothenate 2000 mg, niacinamide 9000 mg, folic acid 370 mg, biotin 15 mg, inositol 10000 mg, MgSO4 6000 mg, ZnSO4 4000 mg, MnSO4 2500 mg, CuSO4 2500 mg, FeSO4 2500 mg, CoSO4 160 mg, Ca(IO3)2 200 mg and Na2SeO3 40 mg.

[0046] The fish meal used in this comparative example was purchased from TASA Fish Product Co., Ltd., Peru; fish oil was purchased from Foshan Damao Feed Co., Ltd.; cellulose content >99.0% was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; methylcellulose was purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.; choline content 50% was purchased from Jujia Biotechnology Co., Ltd.; 2,6-di-tert-butyl-p-cresol content >99.0% was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0047] The preparation method of the comparative feed is as follows: fish meal, corn protein, soy protein concentrate, gluten, flour, vitamin and mineral premix, cellulose, methyl cellulose, sodium alginate, choline and 2,6-di-tert-butyl-p-cresol are all passed through a 60-mesh sieve, weighed and mixed evenly, fish oil and water are added, mixed evenly again, and pellets with a particle size of 1 mm are made using a pelletizer (Tongli Grain Machine, HKJ-218). The pellets are dried in a 60°C forced air drying oven to a moisture content of less than 10%, cooled to room temperature, packed into sealed bags, and stored at -20°C for later use.

[0048] Comparative Example 2

[0049] This comparative example provides a feed, wherein the total fat content of the feed is 128.9 g / kg, the total protein content is 475.8 g / kg, and every 1000 parts by weight of the feed contains the following components in parts by weight: 400 parts of fish meal, 40 parts of corn protein, 150 parts of soybean protein concentrate, 85 parts of gluten, 109.8 parts of flour, 90 parts of fish oil, 10 parts of vitamin and mineral premix, 90 parts of cellulose, 10 parts of methyl cellulose, 10 parts of sodium alginate, 5 parts of choline, and 0.2 parts of 2,6-di-tert-butyl-p-cresol.

[0050] Each kg of vitamin and mineral premix used in this comparative example contains VA 750000 IU, VD3 200000 IU, V E 6000 mg, VK3 2000 mg, VB1 1200 mg, VB2 1200 mg, VB6 1200 mg, VB 12 8 mg, VC21000 mg, d-calcium pantothenate 2000 mg, niacinamide 9000 mg, folic acid 370 mg, biotin 15 mg, inositol 10000 mg, MgSO4 6000 mg, ZnSO4 4000 mg, MnSO4 2500 mg, CuSO4 2500 mg, FeSO4 2500 mg, CoSO4 160 mg, Ca(IO3)2 200 mg and Na2SeO3 40 mg.

[0051] The fish meal used in this comparative example was purchased from TASA Fish Product Co., Ltd., Peru; fish oil was purchased from Foshan Damao Feed Co., Ltd.; cellulose content >99.0% was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; methylcellulose was purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.; choline content 50% was purchased from Jujia Biotechnology Co., Ltd.; 2,6-di-tert-butyl-p-cresol content >99.0% was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0052] The preparation method of the comparative feed is as follows: fish meal, corn protein, soy protein concentrate, gluten, flour, vitamin and mineral premix, cellulose, methyl cellulose, sodium alginate, choline and 2,6-di-tert-butyl-p-cresol are all passed through a 60-mesh sieve, weighed and mixed evenly, fish oil and water are added, mixed evenly again, and pellets with a particle size of 1 mm are made using a pelletizer (HKJ-218, Tongli Grain Machinery). The pellets are dried in a 60°C forced air drying oven to a moisture content of less than 10%, cooled to room temperature, packed into sealed bags, and stored at -20°C for later use.

[0053] Comparative Example 3

[0054] This comparative example provides a feed, wherein the total fat content of the feed is 221.6 g / kg, the total protein content is 474.7 g / kg, and every 1000 parts by weight of the feed contains the following components in parts by weight: 400 parts of fish meal, 40 parts of corn protein, 150 parts of soybean protein concentrate, 85 parts of gluten, 109.8 parts of flour, 180 parts of fish oil, 10 parts of vitamin and mineral premix, 10 parts of methylcellulose, 10 parts of sodium alginate, 5 parts of choline, and 0.2 parts of 2,6-di-tert-butyl-p-cresol.

[0055] Each kg of vitamin and mineral premix used in this comparative example contains VA 750000 IU, VD3 200000 IU, V E 6000 mg, VK3 2000 mg, VB1 1200 mg, VB2 1200 mg, VB6 1200 mg, VB 12 8 mg, VC21000 mg, d-calcium pantothenate 2000 mg, niacinamide 9000 mg, folic acid 370 mg, biotin 15 mg, inositol 10000 mg, MgSO4 6000 mg, ZnSO4 4000 mg, MnSO4 2500 mg, CuSO4 2500 mg, FeSO4 2500 mg, CoSO4 160 mg, Ca(IO3)2 200 mg and Na2SeO3 40 mg.

[0056] The fish meal used in this comparative example was purchased from TASA Fish Product Co., Ltd., Peru; fish oil was purchased from Foshan Damao Feed Co., Ltd.; cellulose content >99.0% was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; methylcellulose was purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.; choline content 50% was purchased from Jujia Biotechnology Co., Ltd.; 2,6-di-tert-butyl-p-cresol content >99.0% was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0057] The preparation method of the comparative feed is as follows: fish meal, corn protein, soy protein concentrate, gluten, flour, vitamin and mineral premix, cellulose, methyl cellulose, sodium alginate, choline and 2,6-di-tert-butyl-p-cresol are all passed through a 60-mesh sieve, weighed and mixed evenly, fish oil and water are added, mixed evenly again, and pellets with a particle size of 1 mm are made using a pelletizer (Tongli Grain Machine, HKJ-218). The pellets are dried in a 60°C forced air drying oven to a moisture content of less than 10%, cooled to room temperature, packed into sealed bags, and stored at -20°C for later use.

[0058] Example 3

[0059] This embodiment provides a method for raising Heilongjiang grayling juveniles.

[0060] Juvenile Heilongjiang graylings were obtained from a farm in Fangzheng County, Harbin City, Heilongjiang Province. After two weeks of acclimation, 900 juveniles of similar size (1.63 ± 0.06) g were randomly divided into 15 stainless steel culture tanks (0.78 m diameter, 0.8 m height, 60 per tank) and provided with recirculating aerated tap water. Juveniles were reared under a 12-h light / 12-h dark photoperiod, with a water temperature of 12.5–13°C, and a daily water exchange rate of 20%. During rearing, the fish were manually fed twice daily at 8:30 and 16:00 until apparent satiety for 8 weeks.

[0061] In order to verify the feeding effect of different groups of feeds, 15 stainless steel water tanks were randomly divided into five groups, with 3 replicates in each group, and each group was fed with the feed provided by Example 1, Example 2, Comparative Example 1, Comparative Example 2 and Comparative Example 3, respectively.

[0062] The feeding effect analysis method and results are as follows:

[0063] 1. Fish growth performance, feed utilization and body index

[0064] Sample Collection: At the end of the feeding experiment, all fish in each tank were counted and weighed. Nine fish were randomly selected from each tank and anesthetized with 0.01 g / L ethyl 3-aminobenzoate sulfonate (MS-222, Sigma-Aldrich) until sedated. Organs and livers were removed and weighed to calculate organ and liver body mass indices. The results are shown in Table 1.

[0065] The differences among the five groups were determined using one-way analysis of variance with Duncan's multivariate range test in SPSS 22.0 statistical software (IBM, USA). P values ​​< 0.05 were considered statistically significant.

[0066] Table 1

[0067]

[0068] As can be seen from Table 1, with the increase of dietary fat level, the weight gain of fish showed an upward trend. The weight gain of fish in the dietary fat level of Example 1, Example 2 and Comparative Example 3 groups was significantly higher than that of fish in Comparative Example 1 and Comparative Example 2 groups (P < 0.05). Protein efficiency, weight gain rate and specific growth rate all increased first and then decreased with the increase of dietary fat level. These parameters were higher in Example 1 group and significantly higher than those in Comparative Example 1 group (P < 0.05). The feed conversion rate of Example 1 group and Comparative Example 3 groups was significantly lower than that of Comparative Example 1 and Comparative Example 2 groups (P < 0.05). The organ-to-body ratio and survival rate of Comparative Example 3 group were significantly higher than those of the other groups (P < 0.05). There were no significant differences in fatness and liver-to-body ratio among the groups (P > 0.05).

[0069] 2. Fish intestinal and liver enzyme activity detection, liver histology detection

[0070] Sample Collection: At the end of the feeding experiment, intestinal and liver samples were collected from three fish per tank, weighed, homogenized with physiological saline, and centrifuged at 2500 rpm for 10 min at 4°C. Digestive and antioxidant enzyme activities were further assayed. Intestinal trypsin, lipase, and amylase activities, as well as intestinal and liver superoxide dismutase, catalase, and malondialdehyde activities, and liver lysozyme, alkaline phosphatase, and acid phosphatase activities were assayed using kits from the Nanjing Jiancheng Bioengineering Research Institute. The results of intestinal and liver enzyme activities in Heilongjiang grayling juveniles are shown in Table 2.

[0071] The fixed livers were transferred to alcohol (70%) and processed for routine histology, stained with hematoxylin and eosin, and examined under a light microscope. Figure 1 .

[0072] Table 2

[0073]

[0074] As can be seen from the results in Table 2, the intestinal trypsin and lipase activities of fish are positively correlated with the fat level in the diet. The intestinal lipase activity of Example 1 group and Comparative Example 3 groups was significantly higher than that of Comparative Example 1 group and Comparative Example 2 group (P < 0.05), and the trypsin activity of Example 1 group and Comparative Example 3 groups was significantly higher than that of the other groups (P < 0.05). There was no significant difference in the intestinal α-amylase content between the groups (P > 0.05). The intestinal superoxide dismutase activity of Example 1 group and Comparative Example 3 groups was significantly higher than that of the other groups (P < 0.05). The intestinal catalase activity of Example 1 group was significantly higher than that of Comparative Example 1 group and Comparative Example 2 group (P < 0.05).

[0075] Different fat levels of diet also significantly affected the activities of superoxide dismutase and catalase in the liver of Heilongjiang grayling (P < 0.05). These parameters were not significantly different between the comparative example 1 group and the comparative example 2 group (P > 0.05), but were significantly different between the example 1 group and the comparative example 3 group (P < 0.05). When the fat content of the diet reached 185.3 g / kg, the malondialdehyde activity in the intestine and liver significantly decreased, and when the fat content of the diet reached 221.6 g / kg, the malondialdehyde activity significantly increased (P < 0.05). When the fat content of the diet reached 185.3 g / kg, the activities of lysozyme, alkaline phosphatase and acid phosphatase in the liver significantly increased (P < 0.05); when the fat content of the diet reached 221.6 g / kg, the activities of lysozyme, alkaline phosphatase and acid phosphatase in the liver significantly decreased (P < 0.05).

[0076] like Figure 1 As shown, no lipid vacuoles were observed in the livers of fish in Comparative Example 1, Comparative Example 2, and Example 2. When the fat content in the feed reached 185.3 g / kg and 221.6 g / kg, a large number of lipid vacuoles were observed in the liver sections, and their abundance and size were positively correlated with the lipid level in the feed.

[0077] 3. Detection of expression levels of lipid metabolism-related genes and immune-related genes in fish liver and intestine

[0078] Sample collection: At the end of the feeding experiment, three fish were collected from each box. Total RNA was extracted from the intestine (n = 6) and liver (n = 6) of fresh fish using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA), and cDNA was synthesized according to the instructions of the PrimeScript™ RT Reagent Kit (Takara, China). Real-time quantitative PCR was performed on an ABI 7500 instrument (Applied Biosystems, USA) using a one-step TB Green® PrimeScript™ RT-PCR Kit (Perfect RealTime) to detect gene expression. Each sample was replicated three times and corrected for β-actin, and then analyzed by 2 −ΔΔCt Methods: The expression levels of target genes were evaluated compared to the control group. Based on the Heilongjiang grayling transcriptome sequence (PRJNA907151), primers specific for PPAR-γ, FAS, CPT1A, TGF-β, NF-κB, and IL-8 were designed using the Primer 3plus (https: / / www.primer3plus.com / ) online tool. The efficiency of these primers was evaluated according to previously reported methods, as shown in Table 3. The primer sequence for β-actin has been previously reported. All primer sequences used in this study are shown in Table 4.

[0079] Table 3

[0080]

[0081] Table 4

[0082]

[0083] Figure 2 This is a comparison of the expression of genes related to lipid metabolism in the liver of Heilongjiang grayling fed with the feed provided in Examples 1 and 2 and Comparative Examples 1, 2, and 3. The columns in each group are Comparative Example 1, Comparative Example 2, Example 2, Example 1, and Comparative Example 3 from left to right; Figure 2As shown, when the dietary fat level in Example 1 was greater than 157.3 g / kg, PPARγ gene expression was significantly lower than that in Comparative Example 1 (P < 0.05). FAS gene expression in Comparative Example 2 was significantly higher than that in Comparative Example 1, and significantly higher than that in Example 1 and Comparative Example 3 (P < 0.05). CPT1A gene expression increased with increasing lipid levels, and CPT1A gene expression was significantly upregulated in Comparative Example 3 compared to the other groups (P < 0.05).

[0084] Figure 3 This is a comparison of the expression of immune-related genes in the liver of Heilongjiang grayling fed with the feed provided in Examples 1 and 2 and Comparative Examples 1, 2, and 3. The columns in each group are Comparative Example 1, Comparative Example 2, Example 2, Example 1, and Comparative Example 3 from left to right; Figure 3 As shown, with the increase in dietary fat level, the NF-κB gene expression level in the comparative example 2 group first increased and then significantly decreased (P < 0.05), while the NF-κB gene expression levels in the example 1 and example 2 groups showed a significant increasing trend (P < 0.05). Compared with the other groups, the TGF-β gene expression level in the comparative example 3 group was significantly increased (P < 0.05). The IL-8 gene expression in the comparative example 3 group was significantly increased compared with the comparative example 1 and comparative example 2 groups, and was significantly increased in the example 1 and example 2 groups compared with the comparative example 1 group (P < 0.05).

[0085] Figure 4 The expression of immune-related genes in the intestine of Heilongjiang grayling juveniles fed with the feed provided in Examples 1 and 2 and Comparative Examples 1, 2, and 3 is compared. The columns in each group are Comparative Example 1, Comparative Example 2, Example 2, Example 1, and Comparative Example 3 from left to right. Figure 4 As shown in the figure, there was no statistically significant difference in NF-κB gene expression among the groups (P > 0.05). TGF-β and IL-8 gene expression increased with increasing dietary fat levels, and was significantly higher in Example 1 and Comparative Example 3 groups than in Comparative Example 1 group (P < 0.05).

[0086] 4. Intestinal Microbial Diversity

[0087] Sample Collection: At the end of the feeding experiment, three fish were collected from each tank, and the intestinal contents of the three fish were pooled into one sample. Total microbial DNA (n = 3) was extracted from the intestinal contents using a commercial DNA extraction kit (MP Biomedicals, Irvine, CA, USA), and the quality was assessed by 1% agarose gel electrophoresis. A library was constructed by amplifying the V3-V4 hypervariable region of bacterial 16S rRNA. The library was sequenced on the Illumina MiSeq platform (Illumina, San Diego, CA, USA).

[0088] Bioinformatic analysis of gut bacteria was performed using the QIIME pipeline (version 1.8.0) to assign qualified reads to operational taxonomic units (OTUs) at a similarity of 97%. Alpha-diversity was assessed using the Sobs, Shannon, ACE, Chao, and Coverage indices. Beta-diversity was analyzed using principal component analysis using the Vegan (Community Ecology) package in R. Unique and shared OTUs between groups were analyzed using Venn diagrams. Differences in gut bacteria between groups were identified at the phylum and genus levels. Linear discriminant analysis effect size (LEfSe) was used to identify genera that differed between the fat levels of the diet. Differences between taxa were analyzed using the Kruskal-Wallis test. P < 0.05 was considered statistically significant.

[0089] A total of 730,531 high-quality reads (3 reads per group) with an average length of 426 bp were obtained from 15 samples. All raw sequencing data were submitted to NCBI (PRJNA1043406). Figure 5 and Figure 6 The comparison chart of Sobs index and Shannon index of intestinal microbial diversity rarefaction curve of Heilongjiang grayling fed with different feed groups is shown in the figure. Figure 5 and Figure 6 It shows that the rarefaction curve tends to saturate, indicating that the raw data can be used for further analysis.

[0090] Table 5 shows the α diversity index of the intestinal microbial community of Heilongjiang grayling juveniles fed with each group of feed.

[0091] Table 5

[0092]

[0093] As shown in Table 5 , there were no statistically significant differences among the treatment groups in the α-diversity analysis of Shannon, Simpson, ACE, Chao1, and coverage indices ( P > 0.05).

[0094] The principal coordinate analysis (PCoA) was used to analyze the β diversity of binary Jaccard and weighted UniFrac indicators at the genus level. Figure 7 and Figure 8 The results of principal coordinate analysis of the intestinal microbial community at the genus level of Heilongjiang grayling fed with each group of feed were plotted based on binary Jaccard indicators and weighted UniFrac metrics. The results showed that the differences between the groups only existed in the binary Jaccard PCoA diagram (P < 0.05).

[0095] Figure 9 Figure 2. Venn diagram analysis results of shared and unique OTUs among intestinal microorganisms of Heilongjiang grayling juveniles fed each group of feed at the genus level. In the Venn diagram analysis, a total of 169 shared OTUs were found in all groups, and the number of OTUs in each group first increased and then decreased with the increase of dietary fat level.

[0096] Figure 10 and Figure 11 The following is a comparison of the relative abundance of intestinal microorganisms at the phylum and genus levels in Heilongjiang grayling juveniles fed with different diets; the results show that the relative abundance of phyla and genera that account for more than 1% of the intestinal microbial community is represented by stacked histograms. Figure 10 As shown, the dominant phyla in all groups were Firmicutes and Actinobacteria. Figure 11 The results showed that at the genus level, the relative abundance of Staphylococcus in comparison group 1 was the most different from that in other groups.

[0097] Figure 12 Figure 2 shows the LEfSe analysis results of the differences in taxonomic abundance of the gut microbiota of Heilongjiang grayling juveniles fed different diets. LEfSe analysis revealed significant differences between 29 taxa. The addition of 185.3 g / kg fat significantly increased the relative abundance of Bacillaceae (g_norank_f_Bacillaceae and g_unclassified_f_ Bacillaceae) and Lactococcus (LDA score >2, P < 0.05).

[0098] Figure 13Bar charts showing the Kruskal-Wallis H test for the genus-level composition of specific microorganisms in the gut microbiota of Heilongjiang grayling juveniles fed each diet group. The length of the histogram represents the abundance of different species. Kruskal-Wallis H tests between groups revealed significant differences (P < 0.05) in 19 genera, including Lactococcus, Bacillus, Pedobacter, Jeotgalicoccus, norank_f_Bacillaceae, and unclassified_f_bacillaceae.

[0099] The Kruskal-Wallis H test was used to further analyze the differences in specific genera such as Lactococcus, Bacillus, Staphylococcus, and Bacillus anthracis among the groups. Figure 14 This is a comparison chart of the relative abundance differences of genera and species among groups of intestinal microorganisms of Heilongjiang grayling fed with different groups of feed using the Kruskal-Wallis H test.

[0100] Figure 14 The results showed that the relative abundance of Staphylococcus and Staphylococcus xylosus in the comparative example 1 group was significantly higher than that in the other groups (P < 0.05); the relative abundance of Bacillus and Bacillus anthracis in the comparative example 1 group decreased with increasing fat levels (P > 0.05); the relative abundance of unclassified_f_ Bacillaceae in the example 1 group first increased and then decreased with increasing fat levels, and was significantly higher than that in the comparative example 2 group (P < 0.05). The relative abundance of Pedobacter first increased, then decreased, and then increased, and the relative abundance of the comparative example 2 group was significantly higher than that in the comparative example 1 group (P < 0.05). The relative abundance of Jeotgalicoccus first decreased, then increased, and then decreased, and the relative abundance of the comparative example 1 group was significantly higher than that of the other groups except the example 1 group (P < 0.05).

[0101] The results show that the Heilongjiang grayling juvenile feed provided by the present invention can improve the specific growth rate and weight gain of Heilongjiang grayling juveniles, improve protein efficiency and reduce feed conversion ratio, increase the activities of intestinal trypsin, lipase, catalase and liver superoxide dismutase, catalase, alkaline phosphatase and acid phosphatase, reduce the liver NF-κB mRNA level, avoid obvious vacuolation of hepatocytes caused by excessive lipid intake, and will not cause a decrease in the abundance of beneficial bacteria and an increase in the abundance of harmful bacteria in the intestine.

Claims

1. A Heilongjiang grayling juvenile feed, characterized in that: The total fat content of the feed is 150-200 g / kg, and the total protein content is 475-477 g / kg. Per 1000 parts by weight of the Heilongjiang grayling juvenile feed, it contains the following components in parts by weight: 400 parts of fish meal, 40 parts of corn protein, 150 parts of soybean protein concentrate, 85 parts of gluten, 109.8 parts of flour, 120-150 parts of fish oil, 10 parts of vitamin and mineral premix, 30-60 parts of cellulose, 10 parts of methyl cellulose, 10 parts of sodium alginate, 5 parts of choline, and 0.2 parts of 2,6-di-tert-butyl-p-cresol.

2. A Heilongjiang grayling juvenile feed according to claim 1, characterized in that The total fat content of the feed is 185.3 g / kg, the total protein content is 476.5 g / kg, and every 1000 parts by weight of the Heilongjiang grayling juvenile feed contains the following components in parts by weight: 400 parts of fish meal, 40 parts of corn protein, 150 parts of soybean protein concentrate, 85 parts of gluten, 109.8 parts of flour, 150 parts of fish oil, 10 parts of vitamin and mineral premix, 30 parts of cellulose, 10 parts of methyl cellulose, 10 parts of sodium alginate, 5 parts of choline, and 0.2 parts of 2,6-di-tert-butyl-p-cresol.

3. A Heilongjiang grayling juvenile feed according to claim 1 or 2, characterized in that, Each kg of the vitamin and mineral premix contains VA 750000 IU, VD3 200000 IU, VE 6000 mg, VK3 2000 mg, VB1 1200 mg, VB2 1200 mg, VB6 1200 mg, VB 12 8 mg, VC 21000 mg, d-calcium pantothenate 2000 mg, niacinamide 9000 mg, folic acid 370 mg, biotin 15 mg, inositol 10000 mg, MgSO4 6000 mg, ZnSO4 4000 mg, MnSO42500 mg, CuSO4 2500 mg, FeSO4 2500 mg, CoSO4 160 mg, Ca(IO3)2 200 mg and Na2SeO3 40 mg.

4. A Heilongjiang grayling juvenile feed according to claim 3, characterized in that, The preparation method of the Heilongjiang grayling juvenile feed comprises the following steps: passing fish meal, corn protein, soybean protein concentrate, gluten, flour, vitamin and mineral premix, cellulose, methyl cellulose, sodium alginate, choline and 2,6-di-tert-butyl-p-cresol through a 60-mesh sieve, weighing and uniformly mixing the mixture, adding fish oil and water, and uniformly mixing the mixture again to prepare a pellet feed, drying the pellet feed to a moisture content of less than 10%, and storing the pellet feed for future use.

5. A method for raising Heilongjiang grayling juveniles, characterized in that: The water temperature for rearing is 12.5-13° C., the dissolved oxygen is maintained at no less than 8 mg / L, the photoperiod is 12 h light / 12 h dark, the water change rate is 20% per day, and the fish are fed a Heilongjiang grayling juvenile feed twice per day until they appear full. The Heilongjiang grayling juvenile feed has a total fat content of 150-200 g / kg and a total protein content of 475-477 g / kg. The Heilongjiang grayling juvenile feed contains the following components in parts by weight per 1000 parts by weight: 400 parts of fish meal, 40 parts of corn gluten, 150 parts of soybean protein concentrate, 85 parts of wheat gluten, 109.8 parts of flour, 120-150 parts of fish oil, 10 parts of a vitamin and mineral premix, 30-60 parts of cellulose, 10 parts of methyl cellulose, 10 parts of sodium alginate, 5 parts of choline, and 0.2 parts of 2,6-di-tert-butyl-p-cresol.

6. A method for raising Heilongjiang grayling juveniles according to claim 5, characterized in that: The Heilongjiang grayling juvenile feed has a total fat content of 185.3 g / kg and a total protein content of 476.5 g / kg. Every 1000 parts by weight of the Heilongjiang grayling juvenile feed contains the following components in parts by weight: 400 parts of fish meal, 40 parts of corn protein, 150 parts of soybean protein concentrate, 85 parts of gluten, 109.8 parts of flour, 150 parts of fish oil, 10 parts of vitamin and mineral premix, 30 parts of cellulose, 10 parts of methyl cellulose, 10 parts of sodium alginate, 5 parts of choline, and 0.2 parts of 2,6-di-tert-butyl-p-cresol.

7. A method for raising Heilongjiang grayling juveniles according to claim 5 or 6, characterized in that: Each kg of the vitamin and mineral premix contains VA 750000 IU, VD3 200000 IU, VE 6000 mg, VK3 2000 mg, VB11200 mg, VB2 1200 mg, VB6 1200 mg, VB 12 8 mg, VC 21000 mg, d-calcium pantothenate 2000 mg, niacinamide 9000 mg, folic acid 370 mg, biotin 15 mg, inositol 10000 mg, MgSO4 6000 mg, ZnSO4 4000 mg, MnSO4 2500 mg, CuSO4 2500 mg, FeSO4 2500 mg, CoSO4 160 mg, Ca(IO3)2 200 mg and Na2SeO340 mg.

8. A method for raising Heilongjiang grayling juveniles according to claim 7, characterized in that: The feeding time is 8:30 and 16:00 every day.

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