Low-carbon feed for reducing body fat and nitrogen and phosphorus emissions of freshwater fish

Abstract

The application discloses a low-carbon feed for reducing the body fat rate and nitrogen and phosphorus discharge of freshwater fish by using Phaeodactylum tricornutum powder, and belongs to the technical field of aquaculture. The low-carbon feed is obtained by replacing part or all of soybean meal protein in the feed with Phaeodactylum tricornutum powder. The Phaeodactylum tricornutum powder can reduce the body fat of freshwater fish by inhibiting fat synthesis related genes and promoting fat decomposition related genes, and can reduce phosphorus discharge and improve the phosphorus retention rate through the AMPK / autophagy pathway. After the Phaeodactylum tricornutum powder is used to replace the soybean meal in the feed, the decomposition of fat and the utilization of nitrogen and phosphorus can be promoted.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of aquaculture, and particularly relates to a low-carbon feed for reducing the fat content and nitrogen and phosphorus emissions of freshwater fish. BACKGROUND

[0002] With the rapid development of the industry, the sustainability of protein sources in aquatic feed and the environmental pollution caused by nitrogen and phosphorus emissions in aquaculture water bodies have become increasingly serious. Anti-nutritional factors such as trypsin inhibitors in traditional feed protein sources (such as soybean meal) can easily cause damage to the intestinal barrier function of fish, reduce the activity of digestive enzymes, and thus lead to low metabolic efficiency of nitrogen and phosphorus in feed, exacerbating the eutrophication of aquaculture water bodies. In addition, the accumulation of liver fat in freshwater fish caused by various factors seriously affects the growth and health of fish. Therefore, exploring new low-carbon protein sources and developing low-carbon feed formulations to reduce the dependence on traditional protein sources and improve abnormal fat accumulation and nitrogen and phosphorus emissions in fish is an important development direction in the current field of aquaculture. SUMMARY

[0003] In view of the above technical problems, the application discloses a low-carbon feed for reducing the fat content and nitrogen and phosphorus emissions of freshwater fish by using Phaeodactylum tricornutum powder.

[0004] The first aspect of the application aims to provide an application of Phaeodactylum tricornutum powder in the preparation of any one of the following three kinds of freshwater fish feed:

[0005] (1) a feed for reducing the fat content of freshwater fish;

[0006] (2) a feed for reducing the nitrogen emissions of freshwater fish;

[0007] (3) a feed for reducing the phosphorus emissions of freshwater fish.

[0008] Microalgae do not occupy arable land resources during cultivation, and the cell wall polysaccharide structure is simple, rich in polysaccharides, proteins, fats, vitamins, pigments and various minerals, which is a potential high-quality green low-carbon feed material. Among them, Phaeodactylum tricornutum belongs to Bacillariophyta, Pennatae, Phaeodactylales and Phaeodactylaceae, and is a marine eukaryotic unicellular microalgae with strong adaptability and rapid growth. The algae are rich in various high-value compounds, with lipid accounting for 20-30% of the dry weight of cells, and eicosapentaenoic acid (EPA) accounting for 30-40% of the total fatty acids; the protein content is 45-50%, which is easily digested by animals, and is a high-quality feed source. Moreover, its high photosynthesis and carbon fixation capacity, high tolerance to various environments and other characteristics make it a potential high-quality green low-carbon protein source. The application of Phaeodactylum tricornutum powder in fish feed raw materials reduces the body fat of freshwater fish by reducing the expression of fish fat synthesis-related genes and increasing the expression of fat decomposition-related genes. Phaeodactylum tricornutum is rich in long-chain polyunsaturated fatty acids (LC-PUFA), especially eicosapentaenoic acid (EPA, content up to 20-30% of total fatty acids), which activates peroxisome proliferator-activated receptor alpha (PPARa) and its downstream genes carnitine palmitoyltransferase 1 (CPT-1), hormone-sensitive lipase (HSL) and adipose triglyceride lipase (ATGL), enhances the fatty acid oxidation capacity in mitochondria and peroxisomes, and reduces the accumulation of triglycerides (TAG) in plasma. By down-regulating the expression of fat synthesis-related genes, including fatty acid synthase (FAS), acetyl-CoA carboxylase (ACC), stearoyl-CoA desaturase 1 (SCD-1) and diacylglycerol acyltransferase 1 (DGAT1), the synthesis of new fat is reduced. The beta-oxidation efficiency in mitochondria and peroxisomes directly affects the energy supply state. When beta-oxidation is enhanced (such as by activating PPARa or supplementing vitamins), fish energy metabolism shifts to lipids, reducing the demand for amino acids for gluconeogenesis, thereby inhibiting the generation of ammonia in the urea cycle and reducing nitrogen excretion.

[0009] Further, the Phaeodactylum tricornutum powder reduces the body fat rate of freshwater fish by reducing the expression of fish fat synthesis-related genes and increasing the expression of fat decomposition-related genes; promotes nitrogen deposition in fish and increases nitrogen retention rate by up-regulating the expression of glutamine synthetase; reduces phosphorus excretion and increases phosphorus retention rate through the AMPK / autophagy pathway.

[0010] Optionally, the freshwater fish includes but is not limited to grass carp, silver carp, bighead carp, crucian carp or common carp.

[0011] Preferably, the method for application is that: the Phaeodactylum tricornutum powder is used to replace part or all of the soybean meal in the basic feed; the replacement rate is 25wt.%-100wt.%.

[0012] More preferably, the method for application is that: the Phaeodactylum tricornutum powder is used to replace part or all of the soybean meal in the basic feed; the replacement rate is 50wt.%.

[0013] The second aspect of the present application aims to provide a low-carbon feed for reducing the body fat and nitrogen and phosphorus emissions of freshwater fish, wherein the Phaeodactylum tricornutum powder is used to replace part or all of the soybean meal in the basic feed of the freshwater fish; and the addition amount of the Phaeodactylum tricornutum powder is 6.19-24.74wt.%.

[0014] Further preferably, in the low-carbon feed for reducing the body fat and nitrogen and phosphorus emissions of freshwater fish, the addition amount of the Phaeodactylum tricornutum powder is 12.37wt.%.

[0015] As preferred, 61.9-247.4 parts of the Phaeodactylum tricornutum powder, 140 parts of rapeseed meal, 180 parts of cottonseed protein, 100 parts of cassava starch, 200 parts of flour, 8-36 parts of soybean oil, 2 parts of choline chloride, 10 parts of premix, 0.5 part of ethoxyquin, 20 parts of calcium dihydrogen phosphate, and 69.6-92.1 parts of rice hull powder are included in 1000 parts of the feed, and the rest is soybean meal.

[0016] As preferred, 123.7 parts of the Phaeodactylum tricornutum powder, 140 parts of rapeseed meal, 180 parts of cottonseed protein, 100 parts of cassava starch, 200 parts of flour, 26 parts of soybean oil, 2 parts of choline chloride, 10 parts of premix, 0.5 part of ethoxyquin, 20 parts of calcium dihydrogen phosphate, and 77.8 parts of rice hull powder are included in 1000 parts of the feed, and the rest is soybean meal.

[0017] Optionally, the content of crude fat in the basic feed is 3.9-4.4%, the content of nitrogen is 5.08-5.41%, the content of phosphorus is 1.03-1.18%, and the content of crude protein is 31.74-33.94%.

[0018] The third aspect of the present application aims to provide a preparation method of a low-carbon feed for reducing the body fat rate and nitrogen and phosphorus emissions of freshwater fish, wherein the raw materials are weighed according to the above-mentioned mass parts, all the raw materials are sequentially mixed and dry-mixed, then granulated, dried, and stored at low temperature.

[0019] Optionally, the drying process is performed in an oven, and the temperature is set to 60℃.

[0020] Optionally, the temperature for low-temperature storage is 4℃.

[0021] The present application has at least the following technical effects:

[0022] The feed for reducing body fat and nitrogen and phosphorus discharge of freshwater fish provided by the present application reduces the expression amount of fish fat synthesis related genes and increases the expression amount of fat decomposition related genes by adding Phaeodactylum tricornutum powder as a feed raw material, thereby reducing the body fat of freshwater fish and reducing nitrogen discharge; by promoting lipid oxidation and activating the AMPK / autophagy pathway to increase the ash content and phosphorus retention rate of the fish body, it has great application potential. BRIEF DESCRIPTION OF DRAWINGS

[0023] The accompanying drawings, which form a part of this application, are included to provide a further understanding of the application and are incorporated in and constitute a part of this application. The embodiments of the present application illustrated in the drawings and their descriptions are used to explain the present application and are not intended to limit the present application. In the drawings:

[0024] Figure 1 The data graph is the growth performance and feed utilization result of PT0 group, PT1 group, PT2 group, PT3 and PT4 group in Example 1 of the present application; SGR: specific growth rate; WGR: weight gain rate; FE: feed efficiency; FR: feed intake rate; *P<0.05 indicates significant difference, and the data is represented as mean ± standard error;

[0025] Figure 2 The data graph is the growth performance and feed utilization result of PT0 group, PT1 group, PT2 group, PT3 and PT4 group in Example 1 of the present application; SGR: specific growth rate; WGR: weight gain rate; FE: feed efficiency; FR: feed intake rate; *P<0.05 indicates significant difference, and the data is represented as mean ± standard error;

[0026] Figure 3 The data graph is the growth performance and feed utilization result of PT0 group, PT1 group, PT2 group, PT3 and PT4 group in Example 1 of the present application; SGR: specific growth rate; WGR: weight gain rate; FE: feed efficiency; FR: feed intake rate; *P<0.05 indicates significant difference, and the data is represented as mean ± standard error;

[0027] Figure 4 The data graph is the growth performance and feed utilization result of PT0 group, PT1 group, PT2 group, PT3 and PT4 group in Example 1 of the present application; SGR: specific growth rate; WGR: weight gain rate; FE: feed efficiency; FR: feed intake rate; *P<0.05 indicates significant difference, and the data is represented as mean ± standard error;

[0028] Figure 5Relative expression levels of grass carp liver lipid decomposition and lipid synthesis related genes in PT0 group, PT1 group, PT2 group, PT3 and PT4 group in Example 1 of the present application; pparα: peroxisome proliferator-activated receptor alpha; cpt1: carnitine palmitoyltransferase 1; hsl: hormone-sensitive lipase; atgl: adipose triglyceride lipase; fas: fatty acid synthase; acc: acetyl-CoA carboxylase 1; scd1: stearoyl-CoA desaturase 1; dgat1: diacylglycerol acyltransferase 1;

[0029] Figure 6 Relative expression levels of grass carp liver nitrogen metabolism related genes in PT0 group, PT1 group, PT2 group, PT3 and PT4 group in Example 1 of the present application; gs: glutamine synthetase; igf1: insulin-like growth factor 1;

[0030] Figure 7 Relative expression levels of grass carp liver phosphorus metabolism related genes in PT0 group, PT1 group, PT2 group, PT3 and PT4 group in Example 1 of the present application; beclin1: benzyl chlorin 1; ampkα1: AMP-dependent protein kinase. DETAILED DESCRIPTION

[0031] Various exemplary embodiments of the present application will now be described in detail, which should be considered to be illustrative of the present application and not restrictive of the same. It should be understood that the detailed description and specific examples, while indicating certain embodiments of the application, are intended to be illustrative only and are not intended to limit the scope of the application.

[0032] It should be understood that the terms used in the present application merely describe particular embodiments and are not intended to limit the present application. In addition, for numerical ranges in the present application, it should be understood that each intermediate value between the upper limit and the lower limit of the range is specifically disclosed. Each smaller range within the larger range is also included in the present application. The upper limit and the lower limit of these smaller ranges can be included or excluded independently.

[0033] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, preferred methods and materials are described. All publications mentioned in this specification are herein incorporated by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any reference in this specification is not intended to constitute an admission that the reference is prior art or that this specification is not entitled to any advantage that can reside in the prior art.

[0034] Many modifications and variations to the illustrative embodiments described herein will be apparent to those of ordinary skill in the art from the foregoing description. Such variations may not depart from the scope of the present application. Other embodiments of the application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. The specification and examples given are exemplary only.

[0035] As used herein, the terms "comprises", "comprising", "includes", "including", "has", "having", "contains", "containing", or variations thereof, are intended to be open-ended terms that mean inclusion, but not limited to, the listed material or step.

[0036] The application discloses application of Phaeodactylum tricornutum powder in preparation of any one of the following three kinds of freshwater fish feed:

[0037] (1) a feed for reducing fat rate of freshwater fish;

[0038] (2) a feed for reducing nitrogen emission of freshwater fish;

[0039] (3) a feed for reducing phosphorus emission of freshwater fish.

[0040] After the application of Phaeodactylum tricornutum powder as a feed raw material, the expression amount of fish fat synthesis related genes is significantly reduced, and the relative expression amount of fat decomposition related genes is significantly increased. After the application of Phaeodactylum tricornutum powder as a feed raw material, the relative expression amount of AMP activated protein kinase (AMPK) and autophagy related gene (Beclin1) is significantly increased. After the application of Phaeodactylum tricornutum powder as a feed raw material, the expression amount of glutamine synthetase (GS) is increased, and the relative expression amount of insulin-like growth factor (IGF1) has no significant influence. After the application of Phaeodactylum tricornutum powder as a feed raw material, the fish plasma triglyceride (TG), total cholesterol (T-CHO), high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C) are significantly decreased. After the application of Phaeodactylum tricornutum powder as a feed raw material, the fish ash is significantly increased. After the application of Phaeodactylum tricornutum powder as a feed raw material, the nitrogen retention rate (NRE) and the phosphorus retention rate (PRE) are significantly increased.

[0041] In the alternative embodiments of the application, the freshwater fish includes but is not limited to grass carp, silver carp, bighead carp, crucian carp or common carp.

[0042] The application method is that Phaeodactylum tricornutum powder is used to replace part or all of soybean meal in the basic feed with equal nitrogen and equal fat; the replacement rate is 25wt.%-100wt.%.

[0043] In the more preferred embodiments, the application method is that Phaeodactylum tricornutum powder is used to replace part or all of soybean meal in the basic feed with equal nitrogen and equal fat; the replacement rate is 50wt.%.

[0044] Based on the above application prospect, the embodiment of the present application also provides a low-carbon feed for reducing body fat and nitrogen and phosphorus emissions of freshwater fish, wherein soybean meal in a basic feed for freshwater fish is partially or totally replaced by Phaeodactylum tricornutum powder; and the addition amount of the Phaeodactylum tricornutum powder is 6.19-24.74 wt.%. After the Phaeodactylum tricornutum powder replaces the soybean meal in the feed, the nitrogen content of the feed is reduced, the phosphorus content of the feed is increased, and the protein of the feed is decreased, which is conducive to the improvement of the nitrogen utilization rate. The accumulation of fat is prone to occur when the phosphorus in the feed is deficient. After the Phaeodactylum tricornutum powder replaces the soybean meal in the feed, the phosphorus content of the feed is increased, which may increase the ash content and the phosphorus retention rate of the fish body by promoting lipid oxidation and activating the AMPK / autophagy pathway.

[0045] In a further preferred embodiment, in the low-carbon feed for reducing body fat and nitrogen and phosphorus emissions of freshwater fish, the addition amount of the Phaeodactylum tricornutum powder is 12.37 wt.%.

[0046] In an optional embodiment, 1000 parts by mass of the feed include 61.9-247.4 parts of the Phaeodactylum tricornutum powder, 140 parts of rapeseed meal, 180 parts of cottonseed protein, 100 parts of cassava starch, 200 parts of flour, 8-36 parts of soybean oil, 2 parts of choline chloride, 10 parts of premix, 0.5 part of ethoxyquin, 20 parts of calcium dihydrogen phosphate, 69.6-92.1 parts of rice husk powder, and the balance is soybean meal.

[0047] In a preferred embodiment, 1000 parts by mass of the feed include 123.7 parts of the Phaeodactylum tricornutum powder, 140 parts of rapeseed meal, 180 parts of cottonseed protein, 100 parts of cassava starch, 200 parts of flour, 26 parts of soybean oil, 2 parts of choline chloride, 10 parts of premix, 0.5 part of ethoxyquin, 20 parts of calcium dihydrogen phosphate, 77.8 parts of rice husk powder, and the balance is soybean meal.

[0048] In an optional embodiment, the content of crude fat in the basic feed is 3.9-4.4%, the content of nitrogen is 5.08-5.41%, the content of phosphorus is 1.03-1.18%, and the content of crude protein is 31.74-33.94%.

[0049] The present application also discloses a preparation method of the low-carbon feed for reducing body fat rate and nitrogen and phosphorus emissions of freshwater fish, wherein the raw materials are weighed according to the above mass parts, all the raw materials are sequentially subjected to dry mixing, then granulation, drying, and low-temperature preservation.

[0050] In an optional embodiment, the drying process is performed in an oven, and the temperature is set to 60℃.

[0051] In an optional embodiment, the temperature for low-temperature preservation is 4℃.

[0052] It should be noted that all the raw materials in the following embodiments are obtained through a conventional commercial purchase route.

[0053] Embodiment 1

[0054] 1. Feed preparation

[0055] Control group feed: The basic fish feed was named PT0, and the specific formula and chemical composition are shown in Table 1.

[0056] Experimental group feed: Replacing the soybean meal in the basic fish feed by 25%, 50%, 75%, and 100%, setting equal nitrogen (30.4%) and equal fat (5.5%) experimental feeds, and named PT1, PT2, PT3, and PT4.

[0057] The feed ingredients were thoroughly mixed and granulated, the granules were dried in an oven at 60°C, and stored in a 4°C environment.

[0058] Table 1 shows the formula and chemical composition of each group of feed

[0059]

[0060]

[0061] Note: The premix was obtained through conventional purchase channels, and the addition amount was consistent in each group. The specific composition had the same effect on nitrogen, phosphorus, protein, and fat in each group.

[0062] 2. Experimental animals and feeding test

[0063] Grass carp was selected as the experimental fish in this example.

[0064] Before the experiment, all fish were acclimated to the control feed for two weeks in an indoor recirculating water culture system. Then, healthy fish of uniform size (initial weight: 65 ± 0.06 g) were randomly assigned to indoor glass tanks (20 fish per tank, 100 x 20 x 50 cm), divided into experimental and control groups, and three parallel treatments were set up for each group.

[0065] The experimental group feed and the control group feed were fed at 8:30 and 16:30 respectively every day, and the culture experiment lasted for 8 weeks. The water temperature was maintained at 28-31°C; the pH value was 7.0-8.5; the dissolved oxygen was 4.6-5.5 mg·L -1 , and the total ammonia nitrogen was <0.1 mg·L -1 .

[0066] 3. Sample collection

[0067] Blood was collected from the caudal vein of six fish using a syringe infiltrated with 0.2% heparin sodium solution. Plasma was obtained by centrifugation at 3000g for 15 min at 4°C, and quickly transferred to -80°C for further analysis. The liver was quickly frozen in liquid nitrogen and stored at -80°C for subsequent enzyme activity and protein gene expression analysis.

[0068] 4. Biochemical analysis

[0069] Chemical composition of feed and fish (crude protein, crude fat, moisture and ash) was determined by AOAC method. Crude protein content was determined by Kjeldahl analysis. Crude fat content was determined using a Soxhlet extractor. Fish body phosphorus content was determined by spectrophotometric method according to GB 5009.87-2016 National Food Safety Standard-Determination of Phosphorus in Foods; fish body total nitrogen content was determined by Kjeldahl method according to GB 5009.5-2016 National Food Safety Standard-Determination of Protein in Foods; feed nitrogen and phosphorus content was determined according to GB / T 6432-2018.

[0070] Plasma biochemical indicators were detected.

[0071] Nitrogen retention rate (NRE) and phosphorus retention rate (PRE) follow the following calculation formula:

[0072] Nitrogen retention efficiency (NRE, %) = 100 x (Wt x CNt - W0 x CN0) / (I x CNf)

[0073] Phosphorus retention efficiency (PRE, %) = 100 x (Wt x CPt - W0 x CP0) / (I x CPf)

[0074] In the formula: I - the amount of feed fed in each net cage at the end of the experiment (g);

[0075] W0, Wt - the body weight of fish in each net cage at the beginning and end of the experiment (g), respectively;

[0076] CN0, CNt - the nitrogen content of fish in each net cage at the beginning and end of the experiment (%), respectively;

[0077] CP0, CPt - the phosphorus content of fish in each net cage at the beginning and end of the experiment (%), respectively;

[0078] CNf, CPf - the nitrogen and phosphorus content of feed in each net cage (%), respectively.

[0079] 5. Real-time quantitative polymerase chain reaction (qRT-PCR)

[0080] Total RNA was extracted from the livers of the control group and each experimental group. Using β-actin as a key gene, qRT-PCR was used to detect relevant indicators, including genes related to fat decomposition, fat synthesis, nitrogen metabolism and phosphorus metabolism.

[0081] 6. Statistical analysis

[0082] The single factor analysis of variance was used to compare the control group and each experimental group by SPSS19.0 software, P<0.05(*) was significant difference, P<0.01(**) was extremely significant difference. All results were expressed as mean ± standard error.

[0083] (II) Analysis of experimental results

[0084] 1. Fish growth performance and body composition

[0085] Figure 1 The effects of feed in the control group and experimental groups on the growth performance and feed utilization of grass carp were shown. From Figure 1 It can be seen that the partial substitution of soybean meal in the basic feed with Phaeodactylum tricornutum powder can significantly improve the feed intake rate (FR), feed efficiency (FE), specific growth rate (SGR) and weight gain rate (WGR) of grass carp.

[0086] The specific calculation method of feed intake rate (FR), feed efficiency (FE), specific growth rate (SGR) and weight gain rate (WGR) is as follows:

[0087] Feed intake rate FR (%BW / d) = 100 x dry matter intake / [number of days x (initial body weight + final body weight) / 2];

[0088] Feed efficiency FE (%) = (100 x total weight gain of fish) / intake;

[0089] Specific growth rate SGR (%) = 100 x [Ln (final body weight) - Ln (initial body weight)] / number of days;

[0090] Weight gain rate WCR (%) = 100 x (final body weight - initial body weight) / initial body weight.

[0091] From Figure 1 It can be seen that the most significant improvement effect is PT2 group.

[0092] Figure 2 The effects of feed in the experimental groups and the control group on the biochemical composition of grass carp were shown. From Figure 2 It can be seen that the application of Phaeodactylum tricornutum powder in the feed can significantly improve the ash content of whole fish of grass carp, and has no significant effect on the protein, fat and moisture of whole fish of grass carp.

[0093] Figure 3 The effects of feed in the experimental groups and the control group on the nitrogen and phosphorus retention rate of grass carp were shown. The results showed that compared with PT0 group, the nitrogen retention rate of PT2, PT3 and PT4 groups and the phosphorus retention rate of PT4 group were significantly improved.

[0094] 2. Plasma biochemical indicators

[0095] Figure 4The effects of experimental and control group feeds on the biochemical indicators of grass carp liver were shown. The results showed that, compared with the control group, the experimental group significantly reduced the plasma triglycerides (TG), total cholesterol (T-CHO), low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HLD-C) of grass carp.

[0096] 3. Basic expression related to liver lipid metabolism

[0097] Figure 5 The effects of experimental and control group feeds on the liver lipid metabolism related genes of grass carp were shown. The results showed that, in terms of lipid decomposition related genes, the relative expression of pparα in PT1, PT2 and PT3 groups was significantly higher than that in PT0 and PT4 groups; compared with PT0 group, the relative expression of cpt1 in PT2 and PT3 groups was significantly increased, and that in PT3 group was significantly higher than that in PT1 group; compared with PT0 group, the relative expression of hsl in PT3 group was significantly increased; compared with PT0 group, the relative expression of atgl in PT2, PT3 and PT4 groups was significantly increased. In terms of lipid synthesis related genes, compared with the control group, the relative expression of fas, acc and scd1 in the algae powder added group was significantly reduced, and the relative expression of dgat1 in PT3 and PT4 groups was significantly lower than that in PT0 and PT1 groups.

[0098] 4. Nitrogen metabolism related gene expression

[0099] Figure 6 The effects of experimental and control group feeds on the nitrogen metabolism related gene expression of grass carp liver were shown. The results showed that the relative expression of gs in PT3 group was significantly increased; there was no significant difference in the relative expression of igf1 among different groups.

[0100] 5. Phosphorus metabolism related gene expression

[0101] Figure 7 The effects of experimental and control group feeds on the phosphorus metabolism related gene expression of grass carp liver were shown. The results showed that, with the increase of the proportion of algae powder replacing soybean meal, the relative expression of beclin1 showed an upward trend, and that in PT3 and PT4 groups was significantly higher than that in PT0 group; the relative expression of ampkα1 in PT2 group was significantly higher than that in other groups.

[0102] From the above experimental results, it can be seen that the application of Phaeodactylum tricornutum powder in feed can reduce the blood lipid indicators and fat synthesis related gene expression of fish, increase the expression of fat decomposition related genes and improve the expression of nitrogen metabolism and phosphorus metabolism related genes.

[0103] Example 2:

[0104] 1. Preparation of feed

[0105] Control group feed: basic fish feed, named control group, the specific formula and chemical composition are shown in Table 2.

[0106] Experimental group feed: Triangular brown Phaeodactylum powder was used to replace soybean meal in the basic fish feed at a proportion of 50%, and experimental feeds with equal nitrogen (30.4%) and equal fat (5.5%) were set.

[0107] The feed preparation process was the same as in Example 1.

[0108] Table 2 is the formula and chemical composition of the feed of each group

[0109]

[0110] 2. Experimental animals and feeding test

[0111] In this example, gibel carp was selected as the experimental fish. The experiment was carried out in outdoor pond net cages, and three parallel treatments were set for each group. Experimental group feed and control group feed were used for apparent satiety feeding at 8:30 and 16:30 respectively every day, and the breeding experiment lasted for 60 days.

[0112] 3. The breeding effect is shown in Table 3.

[0113] Table 3

[0114] Control group Experimental group Effect promotion Initial weight (g) 5.89 5.89 — Final weight (g) 29.33 30.14 2.76％ Feed intake (g / fish) 28.54 28.14 -1.40％ Feed conversion rate (% g body weight / day) 2.7 2.6 -3.7％ Specific growth rate (% / day) 2.68 2.72 1.49％ Feed efficiency (%) 82.13 86.17 4.92％ Protein content of fish at the end of the experiment (%) 14.37 15.18 5.64％ Fat content of fish at the end of the experiment (%) 9.81 8.62 -12.13％ Ash content of fish at the end of the experiment (%) 4.05 4.11 1.48％ Moisture content of fish at the end of the experiment (%) 71.77 72.09 0.45％ Protein deposition rate (%) 38.51 40.2 4.39％ Fat deposition rate (%) 90.15 84.16 -6.64％ Liver-somatic index at the end of the experiment (%) 4.85 5.15 6.19％ Viscera-somatic index at the end of the experiment (%) 14.84 15.48 4.31％ Condition factor at the end of the experiment (%) 3.48 3.98 14.37％ Blood glucose at the end of the experiment (mM) 5.48 5.15 -6.02％ Triglyceride at the end of the experiment (mM) 6.98 5.64 -19.20％ Free fatty acid at the end of the experiment (mEq / L) 0.78 0.48 -38.46％ Cholesterol at the end of the experiment (mM) 8.92 7.48 -16.14％ Nitrogen deposition rate at the end of the experiment (%) 5.51 6.78 23.05％ Phosphorus deposition rate at the end of the experiment (%) 3.76 4.78 27.13％

[0115] As can be seen from Table 3, at the end of the 60-day experiment, the final body weight, feed intake, feed conversion rate, specific growth rate and feed efficiency of the experimental group gibel carp had no significant difference with the control group; the protein content, ash content and moisture content of the fish body of the experimental group had no significant difference with the control group at the end of the experiment, however, the fat content of the fish body of the experimental group (8.62%) was significantly lower than that of the control group (9.81%); the protein deposition rate of the experimental group (40.2%) was higher than that of the control group (38.51%), while the fat deposition rate (84.16%) was lower than that of the control group (90.15%); the plasma triglyceride and free fatty acid content of the experimental group was significantly lower than that of the control group at the end of the experiment; the nitrogen and phosphorus deposition rates of the experimental group (6.78% and 4.78%) were significantly higher than those of the control group (5.51% and 3.76%) at the end of the experiment. Therefore, replacing soybean meal in the basic fish feed with triangular brown Phaeodactylum powder at a proportion of 50% can significantly reduce the body fat of gibel carp, reduce the lipid content such as plasma triglyceride and free fatty acid, and significantly improve the nitrogen and phosphorus retention rate of the fish body, thereby reducing nitrogen and phosphorus emissions.

[0116] Comparative Example 1

[0117] On the basis of Example 2, the same amount of triangular brown Phaeodactylum powder was replaced with Chlorella vulgaris powder.

[0118] The gynogenetic gibel carp was also selected as the experimental fish. The experiment was carried out in outdoor pond net cages, and three parallel treatments were set in each group. The experimental group and the control group were fed at 8:30 and 16:30 every day, respectively, and the feeding experiment lasted for 60 days.

[0119] The culture effect of the present comparative example is shown in Table 4.

[0120] Table 4

[0121]

[0122]

[0123] As can be seen from Table 4, at the end of the 60-day experiment, the terminal body weight, feed intake, feed conversion rate, specific growth rate and feed efficiency of the gynogenetic gibel carp in the experimental group had no significant difference with those in the control group; at the end of the experiment, the fish body fat content (8.56%) had no significant difference with that in the control group (8.24%), and the fat deposition rate and the plasma triglyceride content had no significant difference with those in the control group, indicating that the replacement of feed soybean meal with Chlorella had no significant effect on fat accumulation in gynogenetic gibel carp. And the replacement of feed soybean meal with Chlorella had no significant effect on phosphorus deposition in feed.

[0124] The above is only the preferred specific embodiment of the present application, but the protection scope of the present application is not limited to this, any person skilled in the art can easily think of changes or replacements within the technical range disclosed in the present application, which should be covered in the protection scope of the present application. Therefore, the protection scope of the present application should be subject to the protection scope of the claims.

Claims

1. Application of *Phaeodactylum tricornutum* powder in the preparation of either of the following two freshwater fish feeds: (1) Feed that reduces nitrogen emissions from freshwater fish; (2) Feeds that reduce phosphorus emissions from freshwater fish; Upregulating glutamine synthase expression promotes nitrogen deposition and increases nitrogen retention in fish, or reduces phosphorus excretion and increases phosphorus retention through the AMPK / autophagy pathway.

2. The application according to claim 1, characterized in that, The freshwater fish mentioned are grass carp, silver carp, bighead carp, crucian carp, or common carp.

3. The application according to claim 1, characterized in that, Each 1000 parts by weight of feed contains 61.9–247.4 parts of *Phaeodactylum tricornutum* powder, 140 parts of rapeseed meal, 180 parts of cottonseed protein, 100 parts of cassava starch, 200 parts of wheat flour, 8–36 parts of soybean oil, 2 parts of choline chloride, 10 parts of premix, 0.5 parts of ethoxyquinoline, 20 parts of calcium dihydrogen phosphate, 69.6–92.1 parts of rice husk powder, with the remainder being soybean meal.

4. The application according to claim 3, characterized in that, Each 1000 parts by weight of feed contains 123.7 parts of *Phaeodactylum tricornutum* powder, 140 parts of rapeseed meal, 180 parts of cottonseed protein, 100 parts of cassava starch, 200 parts of wheat flour, 26 parts of soybean oil, 2 parts of choline chloride, 10 parts of premix, 0.5 parts of ethoxyquinoline, 20 parts of calcium dihydrogen phosphate, 77.8 parts of rice husk powder, and the remainder is soybean meal.

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