Application of phenethyl caffeate in improving negative influence of high-carbohydrate feed on growth and metabolism of fishes

Abstract

The invention discloses application of caffeic acid phenethyl ester to improvement of negative effects of high-carbohydrate feed on fish growth and metabolism, and belongs to the technical field of aquaculture. The caffeic acid phenethyl ester is added into a high-carbohydrate feed formula, so that the metabolic capability of adipose tissues, livers and muscles of the grass carp and the weever on glucose can be remarkably enhanced, ectopic deposition of lipid in the livers and the muscles is relieved, fatty liver diseases are improved, muscle protein deposition is promoted, and the immunity of the grass carp and the weever is improved. The inhibiting effect of the high-carbohydrate feed on the growth performance of the grass carp and the weever is reversed. The invention further provides formulas of the high-carbohydrate feed containing the caffeic acid phenethyl ester for the grass carp and the weever respectively, and data support is provided for research on improvement of the utilization capacity of the grass carp and the weever on the high-carbohydrate feed.

Description

Technical Field

[0001] The invention belongs to the technical field of aquaculture, and particularly relates to application of caffeic acid phenethyl ester in improving the negative effects of high-carbohydrate feed on fish growth and metabolism. Background Art

[0002] Grass carp (Ctenopharyngodon idella), as a representative herbivorous fish, is one of the traditional "four major carps" in my country. It has important economic value and is the largest freshwater farmed fish in China. The output in 2023 exceeded 5.9 million tons, an increase of 36,000 tons over 2022. As an emerging farmed species of carnivorous fish, sea bass (Micropterus salmoides) has rapidly emerged in China's aquaculture industry in recent years and is widely regarded as the "fifth major carp". Both grass carp and sea bass have important nutritional value. They are rich in protein, fat, vitamins, unsaturated fatty acids, selenium and minerals, which are beneficial to blood circulation, especially for cardiovascular patients. At the same time, grass carp and sea bass have the advantages of fast growth, tender meat and high economic value. The output has increased rapidly in recent years. Carbohydrates are considered to be the most cost-effective energy source in aquatic feeds, and have the effect of saving protein, which can effectively reduce feed costs. However, compared with mammals, fish have a "congenital diabetic constitution", which leads to a very limited ability of fish to effectively metabolize glucose. This means that both herbivorous grass carp and carnivorous sea bass will suffer from persistent hyperglycemia and hyperlipidemia after ingesting high-carbohydrate feed (HCD), which in turn induces fatty liver, metabolic disorders and growth retardation in grass carp. Therefore, how to enhance the glucose metabolism ability of fish, improve the utilization rate of dietary carbohydrates in farmed fish, and reverse the negative effects of HCD is of great economic significance. Caffeic acid phenethyl ester (CAPE) is a natural flavonoid compound and a key component of propolis. Chinese propolis has the highest content of CAPE, ranging from 15-29 mg / g. A large number of studies have demonstrated the beneficial effects of CAPE, including its anti-inflammatory, antioxidant, antiviral and anti-tumor properties. Mouse studies have shown that CAPE can effectively improve hyperglycemia, glucose tolerance and hyperlipidemia, and reduce the levels of proinflammatory cytokines. In addition, CAPE was found to enhance insulin sensitivity and improve fatty liver in HepG2 cells exposed to high glucose levels. However, it is unclear whether CAPE can alleviate the adverse effects of HCD on fish. Therefore, based on the effects of CAPE, exploring its effects on fish growth and metabolic performance is expected to become an effective nutritional approach to improve the negative effects of HCD use in aquaculture. Summary of the invention

[0003] Technical problem to be solved: In view of the above technical problems, the purpose of the present invention is to disclose the application of caffeic acid phenethyl ester in improving the negative effects of high-carbohydrate feed on fish growth and metabolism, which belongs to the field of aquaculture technology. The present invention is based on the addition of caffeic acid phenethyl ester to the high-carbohydrate feed formula, which can significantly enhance the metabolic capacity of grass carp and seabass adipose tissue, liver and muscle for glucose, alleviate the ectopic deposition of lipids in the liver and muscle, improve fatty liver disease, promote muscle protein deposition, and reverse the inhibitory effect of high-carbohydrate feed on the growth performance of grass carp and seabass. The present invention also provides formulas of high-carbohydrate feeds containing caffeic acid phenethyl ester for grass carp and seabass, respectively, to provide data support for research on improving the ability of grass carp and seabass to utilize high-carbohydrate feeds.

[0004] Technical solution: Application of caffeic acid phenethyl ester in improving the negative effects of high-carbohydrate feed on fish growth and metabolism. Furthermore, the fish include but are not limited to grass carp and sea bass. Furthermore, caffeic acid phenethyl ester is added to the high carbohydrate feed. Furthermore, the added amount of caffeic acid phenethyl ester is 200-800 mg / kg, based on the mass of the high-carbohydrate feed. Furthermore, the formula of the high carbohydrate feed includes a formula of a high carbohydrate feed for grass carp and a formula of a high carbohydrate feed for sea bass. Furthermore, the grass carp high carbohydrate feed formula is composed of: 33% casein, 8% gelatin, 50% corn starch, 5% soybean oil, 0.5% choline chloride, 1% premix, 1% bentonite, 1.4% sodium carboxymethyl cellulose, and 0.01% BHT, calculated by the weight of the high carbohydrate feed. Furthermore, the composition of the high-carbohydrate feed formula for sea bass is: 35% fish meal, 4% soybean meal, 14% chicken meal, 8% soy protein isolate, 8% cottonseed protein, 18% corn starch, 9.22% fish oil, 1% monocalcium phosphate, 1% premix, 0.5% choline chloride, 0.1% vitamin C, 0.5% DL-methionine, 0.1% antioxidant, and 0.58% bentonite, calculated by the weight of the high-carbohydrate feed. Furthermore, the method for preparing the high-carbohydrate feed comprises the following steps: S1. Weigh each component of high carbohydrate feed raw material, stir evenly, and prepare a high carbohydrate feed base; S2. dissolving caffeic acid phenethyl ester in water to obtain a caffeic acid phenethyl ester solution; S3. The caffeic acid phenethyl ester solution is thoroughly mixed with the base material to prepare a high carbohydrate feed premix; S4. pelletizing the high carbohydrate feed premix to obtain pellet feed; S5. The pellet feed is dried and packaged and stored at -20°C to obtain a high carbohydrate feed. Furthermore, the amount of water added in step S2 is 30-50% of the high carbohydrate feed. Beneficial effects: 1. In the application of caffeic acid phenethyl ester provided by the present invention in improving the ability of grass carp and sea bass to utilize high-carbohydrate feed, caffeic acid phenethyl ester effectively promotes the mRNA expression of glut4, gk and pk in the adipose tissue of grass carp and sea bass, and inhibits the mRNA expression of pdk4, thereby significantly improving the glucose metabolism ability of the adipose tissue of grass carp and sea bass, while reducing the serum glucose and serum triglyceride levels, improving serum biochemical indicators, and alleviating the negative impact of a high-carbohydrate diet on health. 2. In the application of the caffeic acid phenethyl ester provided by the present invention in improving the ability of grass carp and sea bass to utilize high-carbohydrate feeds, the caffeic acid phenethyl ester can significantly reduce the triglyceride content in the adipose tissue, liver and muscle of grass carp, alleviate the ectopic deposition of lipids in the liver and muscle, alleviate lipid accumulation, and improve fatty liver disease, thereby improving the ability of grass carp and sea bass to utilize high-carbohydrate feeds and reversing the inhibitory effect of high-carbohydrate feeds on the growth performance of grass carp and sea bass. 3. The present invention provides a high-carbohydrate feed formula with added caffeic acid phenethyl ester, which can not only improve the utilization ability of fish to high-carbohydrate feed, but also improve the growth performance and protein content of fish, and give full play to the protein-saving effect of carbohydrates. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 Effects of feed prepared in Example 1, Comparative Example 1 and Comparative Example 2 on serum glucose content and TG content of grass carp, wherein (A) serum glucose content, (B) serum TG content; Figure 2 The effects of feeding the feed prepared in Example 1, Comparative Example 1 and Comparative Example 2 on adipose tissue lipid accumulation and sugar metabolism genes of grass carp, including (A) grass carp abdominal fat coefficient, (B) grass carp adipose tissue TG content, (C) adipose tissue sugar metabolism-related gene expression, including glut4, gk, pk, pdk4; Figure 3 Effects of feeding the feed prepared in Example 1, Comparative Example 1 and Comparative Example 2 on liver lipid accumulation and glucose metabolism genes of grass carp, including (A) liver-to-body ratio, (B) grass carp liver TG content, (C) liver glucose metabolism-related gene expression, including glut4, gk, pk, pdk4; Figure 4The effects of feeding the feed prepared in Example 1, Comparative Example 1 and Comparative Example 2 on muscle lipid accumulation and sugar metabolism genes of grass carp, including (A) grass carp muscle TG content, (B) muscle sugar metabolism-related gene expression, including glut4, gk, pk, pdk4; Figure 5 Effects of feeding the feed prepared in Example 2, Comparative Example 3 and Comparative Example 4 on the glucose content and TG content in the serum of sea bass, wherein (A) the glucose content in the serum, (B) the TG content in the serum; Figure 6 Effects of feeding the feed prepared in Example 2, Comparative Example 3 and Comparative Example 4 on lipid accumulation in adipose tissue and glucose metabolism genes of seabass, including (A) abdominal fat coefficient of seabass, (B) TG content in adipose tissue of seabass, and (C) expression of genes related to glucose metabolism in adipose tissue, including glut4, gk, pk, and pdk4; Figure 7 Effects of feeding the feed prepared in Example 2, Comparative Example 3 and Comparative Example 4 on liver lipid accumulation and sugar metabolism genes of seabass, including (A) liver-to-body ratio, (B) TG content in seabass liver, and (C) expression of genes related to liver sugar metabolism, including glut4, gk, pk, and pdk4; Figure 8 The effects of feeding the feed prepared in Example 2, Comparative Example 3 and Comparative Example 4 on muscle lipid accumulation and sugar metabolism genes of seabass are studied, including (A) TG content in seabass muscle, and (B) expression of sugar metabolism-related genes in muscle, including glut4, gk, pk, and pdk4. DETAILED DESCRIPTION The present invention will be further described below in conjunction with embodiments, which are explanations of the present invention and are not limited to the following embodiments: The raw materials used in the present invention: fish meal, soybean meal, chicken meal, soy protein isolate, cottonseed protein, corn starch, fish oil, calcium dihydrogen phosphate, premix, choline chloride, vitamins, DL-methionine, antioxidant, bentonite, casein, gelatin, corn starch, soybean oil, choline chloride, premix, microcrystalline cellulose, BHT, are all purchased from Huaqin Agriculture and Animal Husbandry Technology Co., Ltd.; caffeic acid phenethyl ester is purchased from Shanghai MCE Company. Example 1 A method for preparing a high-carbohydrate feed for grass carp supplemented with caffeic acid phenethyl ester comprises the following steps: S1. Weigh 330g casein, 80g gelatin, 500g corn starch, 50g soybean oil, 5g choline chloride, 10g premix, 13.8g microcrystalline cellulose, 1g BHT, stir well to prepare a high carbohydrate feed base; S2. Dissolving 800 mg of caffeic acid phenethyl ester in 400 mL of water to obtain a caffeic acid phenethyl ester solution; S3. The caffeic acid phenethyl ester solution is thoroughly mixed with the base material to prepare a high carbohydrate feed premix; S4. pelletizing the high carbohydrate feed premix to obtain pellet feed; S5. The pellet feed is dried and packaged, and stored at -20°C to obtain a high-carbohydrate feed for grass carp. Comparative Example 1 The difference between this comparative example and Example 1 is that caffeic acid phenethyl ester is not added. A method for preparing a grass carp high-carbohydrate feed comprises the following steps: S1. Weigh 330g casein, 80g gelatin, 500g corn starch, 50g soybean oil, 5g choline chloride, 10g premix, 14g microcrystalline cellulose, 1g BHT, stir well to prepare a high carbohydrate feed base; S4. pelletizing the high carbohydrate feed base to obtain pellet feed; S5. The pellet feed is dried and packaged, and stored at -20°C to obtain a high-carbohydrate feed for grass carp. Comparative Example 2 The difference between this comparative example and Example 1 is that caffeic acid phenethyl ester is not added, and the added amount of corn starch is 30%, and the added amount of microcrystalline cellulose is 21.4%. A method for preparing grass carp feed comprises the following steps: S1. Weigh 330g casein, 80g gelatin, 300g corn starch, 50g soybean oil, 5g choline chloride, 10g premix, 214g microcrystalline cellulose, 1g BHT, stir well to prepare grass carp feed base; S4. The grass carp feed base is granulated to obtain pellet feed; S5. The pellet feed is dried and packaged, and stored at -20°C to obtain grass carp feed. Grass carp performance test The feed prepared in Example 1, Comparative Example 1 and Comparative Example 2 was used to feed grass carp, and 180 grass carp fry with an initial body weight of 12.69±0.05g were selected. The test fish were provided by the Ankang Fishery Experimental Demonstration Station of Northwest Agriculture and Forestry University. The test fish were randomly divided into three treatment groups, each with three repeated cages, and 20 fish in each cage. They were temporarily raised for two weeks before the start of the formal experiment. After the start of the experiment, the breeding lasted for 56 days, with a total daily food intake of 2% of the initial body weight, and fed 3 times a day (08:00, 12:00 and 17:00); during the entire feeding experiment, the water temperature was maintained in the range of 23-28°C, the dissolved oxygen level varied between 7-11mg / L, the pH value was in the range of 7.5-8, and the ammonia nitrogen concentration was maintained below 0.05mg / L. (1) Growth performance indicators After the test, the fish were anesthetized by soaking in a 100 mg / L MS-222 solution, and then the growth performance indicators of grass carp were calculated and recorded as follows: Weight gain rate (WGR, %) = 100 × (final weight initial weight) / initial weight; Specific growth rate (SGR, % / d) = 100 × (ln (final weight) - ln (initial weight)) / culture days; Protein deposition rate (PDR) = (protein increment) / (protein intake); Feed conversion ratio (FCR) = feed dry weight / (final weight-initial weight); Table 1 Growth performance indicators of Example 1, Comparative Example 1 and Comparative Example 2 Growth performance indicators Comparative Example 2 Comparative Example 1 Example 1 Weight (g) <![CDATA[24.57±0.26 a ]]> <![CDATA[21.86±0.31 b ]]> <![CDATA[24.40±0.27 a ]]> Weight gain rate (WGR, %) <![CDATA[79.40±1.60 a ]]> <![CDATA[59.88±2.06 b ]]> <![CDATA[78.44±2.42 a ]]> Specific growth rate (SGR, %) <![CDATA[1.03±0.03 a ]]> <![CDATA[0.84±0.02 b ]]> <![CDATA[1.03±0.02 a ]]> Whole fish crude protein (%) <![CDATA[29.91±0.50 a ]]> <![CDATA[19.27±0.74 b ]]> <![CDATA[30.05±0.92 a ]]> Protein deposition rate (PDR, %) <![CDATA[14.63±0.07 a ]]> <![CDATA[13.80±0.11 b ]]> <![CDATA[14.72±0.12 a ]]> Feed Coefficient (FCR) <![CDATA[1.42±0.02 b ]]> <![CDATA[1.88±0.07 a ]]> <![CDATA[1.43±0.05 b ]]> As shown in Table 1, compared with Comparative Example 1, after feeding the feed prepared in Example 1 to grass carp, the weight, weight gain rate and specific growth rate of grass carp increased significantly, but the feed coefficient decreased significantly. Comparative Example 2 is a grass carp feed with normal carbohydrate levels, while Example 1 is a high carbohydrate feed with added caffeic acid phenethyl ester. There is no significant difference in the levels of weight, weight gain rate, specific growth rate, whole fish crude protein, protein deposition rate and feed coefficient between Example 1 and Comparative Example 2, indicating that the addition of caffeic acid phenethyl ester to high carbohydrate feed can significantly reverse the negative effect of high carbohydrate feed on crude protein of grass carp and effectively improve the growth of grass carp. (2) Determination of glucose and triglyceride levels in serum Blood samples were collected from three groups of grass carp, and serum triglyceride (TG, mmol / L) and glucose (GLU, mmol / L) were analyzed using BK-400 automatic biochemical analyzer. Depend on Figure 1 It can be seen that the high-carbohydrate feed prepared in Comparative Example 1 can significantly increase the content of triglycerides and glucose in the serum of grass carp. The high carbohydrate level can induce the accumulation of glucose and TG in the serum of grass carp, and induce hyperglycemia and hyperlipidemia. However, the experimental results show that the glucose content and triglyceride content in the serum of grass carp fed with the high-carbohydrate feed added with caffeic acid phenethyl ester in Example 1 are significantly reduced, and are similar to the feeding results of the feed with normal carbohydrate levels in Comparative Example 2, indicating that the addition of caffeic acid phenethyl ester can reduce the content of triglycerides and glucose in the serum of grass carp fed with high-carbohydrate feed, and restore to normal levels, which is helpful to reduce the adverse effects of high-carbohydrate feed on grass carp. (3) Hepatic body index and intraperitoneal fat index Six fish were randomly selected from each cage of the three treatment groups for measurement of hepatosomatic index and intraperitoneal fat index, which were calculated as follows: Intraperitoneal fat index (IPFI, %) = 100 × abdominal fat weight / final weight; Liver-to-body ratio (HSI, %) = 100 × liver weight / final weight. (4) Tissue triglyceride content The triglyceride content in liver, muscle and adipose tissue was measured using a tissue TG assay kit (E1013, Beijing Pulilai Gene Technology Co., Ltd., Beijing, China). (5) Gene expression The liver, intraperitoneal fat tissue and muscle tissue of the experimental fish from the three treatment groups (9 fish per cage) were carefully separated on ice, and each tissue sample was placed in a cryovial and stored in an ultra-low temperature freezer at -80°C for further analysis. Gene expression measurement: RNA was extracted from adipose tissue, liver and muscle and reverse transcribed into cDNA. Then, real-time fluorescence quantitative PCR was performed with β-actin as internal reference and 2 -ΔΔCT Methods Quantitative analysis was performed to determine the mRNA expression of glucose metabolism-related genes glut4, gk, pk and pdk4 in grass carp tissues. Data analysis: Data are presented as mean ± standard deviation (mean ± SE). All data were tested for normality and homogeneity by one-way analysis of variance using SPSS 22.0. Post hoc Tukey's test was used to determine the differences between treatments, and the significance level was set at p < 0.05. Glucose transporter 4 (glut4) plays a vital role in maintaining the dynamic balance of glucose in the body. Glucokinase (gk) is the first key enzyme in intracellular glucose metabolism and plays a core role in the regulation of blood glucose homeostasis. Pyruvate kinase (pk) is one of the main rate-limiting enzymes in glycolysis and an important enzyme in glucose metabolism. Pyruvate dehydrogenase kinase 4 (pdk4) is one of the key enzymes regulating the transition from mitochondrial oxidative phosphorylation to glycolysis and plays an important role in glucose and lipid metabolism and ATP formation. Depend on Figure 2It can be seen that compared with Comparative Example 1, the feed prepared in Example 1 can significantly reduce the abdominal fat index and adipose tissue TG content of grass carp, alleviate the accumulation of abdominal fat lipids in grass carp, and at the same time, the mRNA expression levels of glut4, gk and pk in the adipose tissue of grass carp are significantly higher than those in Comparative Example 1 and Comparative Example 2, while the mRNA expression level of pdk4 is lower than that in Comparative Example 1, indicating that caffeic acid phenethyl ester can effectively promote the expression of glut4, gk and pk genes in the adipose tissue of grass carp, significantly inhibit the gene expression of pdk4, improve the metabolic capacity of adipose tissue of grass carp for glucose, and further improve the utilization of high-carbohydrate feed by grass carp. Depend on Figure 3 It can be seen that Example 1 is a high carbohydrate with added caffeic acid phenethyl ester. When it is used to feed grass carp, the liver-to-body ratio and liver TG content in the liver tissue of grass carp are significantly lower than those in Comparative Example 1, and there is no significant difference with Comparative Example 2 (normal carbohydrate level feed), indicating that caffeic acid phenethyl ester can significantly improve the effect of high carbohydrate feed on the liver-to-body ratio and liver TG of grass carp; in Comparative Example 1, the mRNA expression levels of glut4, gk and pk in the liver tissue of grass carp are significantly inhibited, while the mRNA expression level of pdk4 is promoted, while the mRNA expression level of glut4, gk, pk and pdk4 in the liver tissue of grass carp in Example 1 returns to normal, indicating that caffeic acid phenethyl ester can effectively reverse the inhibitory effect of high carbohydrate feed on the glucose metabolism ability of grass carp liver. Depend on Figure 4 It can be seen that in Example 1, high carbohydrates to which caffeic acid phenethyl ester is added are used to feed grass carp. The TG content in grass carp muscle tissue is significantly lower than that in Comparative Example 1, but there is no significant difference with Comparative Example 2 (normal carbohydrate level feed), indicating that caffeic acid phenethyl ester can significantly improve the lipid content in grass carp muscle and reduce the TG level in muscle; there is no significant difference between Example 1 and Comparative Example 2 (normal carbohydrate level feed) in the mRNA expression levels of glut4, gk, pk and pdk4 in grass carp muscle tissue, while Comparative Example 1 shows a significant inhibition level for the mRNA expression levels of glut4, gk and pk in grass carp muscle tissue, while the mRNA expression level of pdk4 shows a significant promotion effect, indicating that caffeic acid phenethyl ester can promote the expression of glut4, gk and pk in grass carp muscle tissue, and inhibit the expression of pdk4, effectively reversing the negative effect of high carbohydrate feed on the glucose metabolism capacity of grass carp muscle. Example 2 A method for preparing a high-carbohydrate feed for sea bass supplemented with caffeic acid phenethyl ester comprises the following steps: S1. Weigh 350g fish meal, 40g soybean meal, 140g chicken meal, 80g soy protein isolate, 80g cottonseed protein, 180g corn starch, 92.2g fish oil, 10g calcium dihydrogen phosphate, 10g premix, 5g choline chloride, 1g vitamin C, 5g DL-methionine, 1g antioxidant, 5.8g bentonite, stir well to prepare a high carbohydrate feed base; S2. Dissolving 800 mg of caffeic acid phenethyl ester in 400 mL of water to obtain a caffeic acid phenethyl ester solution; S3. The caffeic acid phenethyl ester solution is thoroughly mixed with the base material to prepare a high carbohydrate feed premix; S4. pelletizing the high carbohydrate feed premix to obtain pellet feed; S5. The pellet feed is dried and packaged, and stored at -20°C to obtain a high-carbohydrate feed for sea bass. Comparative Example 3 The difference between this comparative example and Example 2 is that caffeic acid phenethyl ester is not added. A method for preparing a high-carbohydrate feed for sea bass comprises the following steps: S1. Weigh 350g fish meal, 40g soybean meal, 140g chicken meal, 80g soy protein isolate, 80g cottonseed protein, 180g corn starch, 92.2g fish oil, 10g calcium dihydrogen phosphate, 10g premix, 5g choline chloride, 1g vitamin C, 5g DL-methionine, 1g antioxidant, 5.8g bentonite, stir well to obtain a high-carbohydrate feed base for sea bass; S2. pelletizing the high carbohydrate feed base to obtain pellet feed; S3. The pellet feed is dried and packaged, and stored at -20°C to obtain a high-carbohydrate feed for sea bass. Comparative Example 4 The difference between this comparative example and Example 2 is that caffeic acid phenethyl ester is not added, and the added amount of corn starch is 8%, and the added amount of bentonite is 10.58%. A method for preparing seabass feed comprises the following steps: S1. Weigh 350g fish meal, 40g soybean meal, 140g chicken meal, 80g soy protein isolate, 80g cottonseed protein, 80g corn starch, 92.2g fish oil, 10g calcium dihydrogen phosphate, 10g premix, 5g choline chloride, 1g vitamin C, 5g DL-methionine, 1g antioxidant, 105.8g bentonite, stir well to obtain a perch feed base; S2. pelletizing the high carbohydrate feed base to obtain pellet feed; S3. The pellet feed is dried and packaged, and stored at -20°C to obtain sea bass feed. Perch Performance Test Feeds prepared in Example 2, Comparative Example 3 and Comparative Example 4 were used to feed sea bass, and 180 sea bass fry with an initial body weight of 122.54±0.58g were selected. The test fish were provided by the Ankang Fishery Experimental Demonstration Station of Northwest Agriculture and Forestry University. The test fish were randomly divided into three treatment groups, each with three repeated cages, and 20 fish in each cage. They were temporarily raised for two weeks before the start of the formal experiment. After the start of the experiment, the breeding lasted for 56 days, with a total daily food intake of 2% of the initial body weight, and fed 3 times a day (08:00, 12:00 and 17:00); during the entire feeding experiment, the water temperature was maintained in the range of 24°C to 26°C, the dissolved oxygen level was above 6mg / L, the pH value was in the range of 7.5-8, and the ammonia nitrogen concentration was maintained below 0.03mg / L. (1) Growth performance indicators After the test, the fish were anesthetized by immersion in a 100 mg / L MS-222 solution, and then the growth performance indicators of the sea bass were calculated and recorded as follows: Weight gain rate (WGR, %) = 100 × (final weight initial weight) / initial weight; Specific growth rate (SGR, % / d) = 100 × (ln (final weight) - ln (initial weight)) / culture days; Protein deposition rate (PDR) = (protein increment) / (protein intake); Feed conversion ratio (FCR) = feed dry weight / (final weight-initial weight); Table 2 Growth performance indicators of Example 2, Comparative Example 3 and Comparative Example 4 As shown in Table 2, compared with Comparative Example 3, after feeding the feed prepared in Implementation 2 to the seabass, the weight, weight gain rate, specific growth rate and protein deposition rate of the seabass were significantly increased, but the feed coefficient was significantly reduced. Comparative Example 4 is a seabass feed with normal carbohydrate levels, while Example 2 is a high-carbohydrate feed with added caffeic acid phenethyl ester. There is no significant difference in the levels of weight, weight gain rate, specific growth rate, whole fish crude protein and feed coefficient between Example 2 and Comparative Example 4, indicating that the addition of caffeic acid phenethyl ester to high-carbohydrate feed can significantly reverse the negative effect of high-carbohydrate feed on crude protein of seabass and effectively improve the growth of seabass. (2) Determination of glucose and triglyceride levels in serum Blood samples were collected from three groups of seabass, and serum triglyceride (TG, mmol / L) and glucose (GLU, mmol / L) were analyzed using BK-400 automatic biochemical analyzer. Depend on Figure 5It can be seen that feeding the high-carbohydrate feed prepared in Comparative Example 3 with seabass can significantly increase the content of triglycerides and glucose in the serum of seabass. The high carbohydrate level can induce the accumulation of glucose and TG in the serum of seabass, and induce hyperglycemia and hyperlipidemia. However, the experimental results show that the glucose content and triglyceride content in the serum of seabass fed with the high-carbohydrate feed added with caffeic acid phenethyl ester in Example 2 are significantly reduced, and are similar to the feeding results of the feed with normal carbohydrate levels in Comparative Example 4, indicating that the addition of caffeic acid phenethyl ester can reduce the content of triglycerides and glucose in the serum of seabass fed with high-carbohydrate feed, and restore them to normal levels, which helps to reduce the adverse effects of high-carbohydrate feed on seabass. (3) Hepatic body index and intraperitoneal fat index Six fish were randomly selected from each cage of the three treatment groups for measurement of hepatosomatic index and intraperitoneal fat index, which were calculated as follows: Intraperitoneal fat index (IPFI, %) = 100 × abdominal fat weight / final weight; Liver-to-body ratio (HSI, %) = 100 × liver weight / final weight. (4) Tissue triglyceride content The triglyceride content in liver, muscle and adipose tissue was measured using a tissue TG assay kit (E1013, Beijing Pulilai Gene Technology Co., Ltd., Beijing, China). (5) Gene expression The liver, intraperitoneal fat tissue and muscle tissue of the experimental fish from the three treatment groups (9 fish per cage) were carefully separated on ice, and each tissue sample was placed in a cryovial and stored in an ultra-low temperature freezer at -80°C for further analysis. Gene expression measurement: RNA was extracted from adipose tissue, liver and muscle and reverse transcribed into cDNA. Then, real-time fluorescence quantitative PCR was performed with β-actin as internal reference and 2 -ΔΔCT Methods Quantitative analysis was performed to determine the mRNA expression of glucose metabolism-related genes glut4, gk, pk and pdk4 in grass carp tissues. Data analysis: Data are presented as mean ± standard deviation (mean ± SE). All data were tested for normality and homogeneity by one-way analysis of variance using SPSS 22.0. Post hoc Tukey's test was used to determine the differences between treatments, and the significance level was set at p < 0.05. Glucose transporter 4 (glut4) plays a vital role in maintaining the dynamic balance of glucose in the body. Glucokinase (gk) is the first key enzyme in intracellular glucose metabolism and plays a core role in the regulation of blood glucose homeostasis. Pyruvate kinase (pk) is one of the main rate-limiting enzymes in glycolysis and an important enzyme in glucose metabolism. Pyruvate dehydrogenase kinase 4 (pdk4) is one of the key enzymes regulating the transition from mitochondrial oxidative phosphorylation to glycolysis and plays an important role in glucose and lipid metabolism and ATP formation. Depend on Figure 6 It can be seen that compared with Comparative Example 3, the feed prepared in Example 2 can significantly reduce the abdominal fat index and adipose tissue TG content of seabass, alleviate the accumulation of abdominal fat lipids in seabass, and at the same time, the mRNA expression levels of glut4, gk and pk in the adipose tissue of seabass are significantly higher than those in Comparative Example 3 and Comparative Example 4, while the mRNA expression level of pdk4 is lower than that in Comparative Example 3, indicating that caffeic acid phenethyl ester can effectively promote the expression of glut4, gk and pk genes in adipose tissue of seabass, significantly inhibit the gene expression of pdk4, improve the metabolic capacity of adipose tissue of seabass for glucose, and further improve the utilization of high-carbohydrate feed by seabass. Depend on Figure 7 It can be seen that Example 2 is a high-carbohydrate feed added with caffeic acid phenethyl ester. When used to feed seabass, the TG content in the liver tissue of seabass was significantly lower than that in Comparative Example 3, and there was no significant difference with Comparative Example 4 (normal carbohydrate level feed), indicating that caffeic acid phenethyl ester can significantly improve the effect of high-carbohydrate feed on seabass liver TG; Comparative Example 3 significantly inhibited the mRNA expression levels of glut4, gk and pk in seabass liver tissue, and promoted the mRNA expression level of pdk4, while in Example 2, the mRNA expression levels of glut4, gk, pk and pdk4 in seabass liver tissue returned to normal, indicating that caffeic acid phenethyl ester can effectively reverse the inhibitory effect of high-carbohydrate feed on the glucose metabolism capacity of seabass liver. Depend on Figure 8It can be seen that in Example 2, a high-carbohydrate feed to which caffeic acid phenethyl ester is added is used to feed seabass. The TG content in the muscle tissue of seabass is significantly lower than that in Comparative Example 3, but there is no significant difference with Comparative Example 4 (normal carbohydrate level feed), indicating that caffeic acid phenethyl ester can significantly improve the lipid content in seabass muscle and reduce the TG level in muscle; there is no significant difference between Example 2 and Comparative Example 4 (normal carbohydrate level feed) in the mRNA expression levels of glut4, gk, pk and pdk4 in seabass muscle tissue, while Comparative Example 3 shows a significant inhibition level for the mRNA expression levels of glut4, gk and pk in seabass muscle tissue, while the mRNA expression level of pdk4 shows a significant promotion effect, indicating that caffeic acid phenethyl ester can promote the expression of glut4, gk and pk in seabass muscle tissue, and inhibit the expression of pdk4, effectively reversing the negative effect of high-carbohydrate feed on the glucose metabolism capacity of seabass muscle. The above is only a preferred embodiment of the present invention and does not limit the present invention in any form. Any technician familiar with the art can make many possible changes and modifications to the technical solution of the present invention by using the above disclosed methods and technical contents without departing from the spirit and technical solution of the present invention, or modify it into an equivalent embodiment of equivalent changes. Therefore, any simple modification, equivalent replacement, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention still falls within the scope of protection of the technical solution of the present invention.

Claims

1. Application of caffeic acid phenethyl ester in improving the negative effects of high carbohydrate feed on fish growth and metabolism.

2. The use according to claim 1, characterized in that: The fish include but are not limited to grass carp and sea bass.

3. The use according to claim 1, characterized in that: Caffeic acid phenethyl ester is added to the high carbohydrate feed.

4. The use according to claim 3, characterized in that: The added amount of the caffeic acid phenethyl ester is 200-800 mg / kg, calculated based on the mass of the high-carbohydrate feed.

5. The use according to claim 1, characterized in that: The formula of the high carbohydrate feed includes a grass carp high carbohydrate feed formula and a sea bass high carbohydrate feed formula.

6. The use according to claim 5, characterized in that: The grass carp high-carbohydrate feed formula comprises: 33% casein, 8% gelatin, 50% corn starch, 5% soybean oil, 0.5% choline chloride, 1% premix, 1% bentonite, 1.4% sodium carboxymethyl cellulose and 0.01% BHT, calculated by the weight of the high-carbohydrate feed.

7. The use according to claim 5, characterized in that: The high-carbohydrate feed formula for sea bass comprises: 35% fish meal, 4% soybean meal, 14% chicken meal, 8% soybean protein isolate, 8% cottonseed protein, 18% corn starch, 9.22% fish oil, 1% monocalcium phosphate, 1% premix, 0.5% choline chloride, 0.1% vitamin C, 0.5% DL-methionine, 0.1% antioxidant and 0.58% bentonite, calculated by the weight of the high-carbohydrate feed.

8. The use according to any one of claims 1 to 7, characterized in that: The method for preparing the high-carbohydrate feed comprises the following steps: S1. Weigh each component of high carbohydrate feed raw material, stir evenly, and prepare a high carbohydrate feed base; S2. dissolving caffeic acid phenethyl ester in water to obtain a caffeic acid phenethyl ester solution; S3. The caffeic acid phenethyl ester solution is thoroughly mixed with the base material to prepare a high carbohydrate feed premix; S4. pelletizing the high carbohydrate feed premix to obtain pellet feed; S5. The pellet feed is dried and packaged and stored at -20°C to obtain a high carbohydrate feed.

9. The use according to claim 8, characterized in that: The amount of water added in step S2 is 30-50% of the high carbohydrate feed.

Images