Feed for enhancing heavy metal cadmium stress resistance of juvenile carp

Abstract

The invention discloses a feed for enhancing heavy metal cadmium stress resistance of juvenile carp. The feed is prepared from the following raw materials in percentage by weight: 8% of fish meal, 5% of chicken meal, 30% of soybean meal, 4% of chlorella, 6.5% of cottonseed protein concentrate, 4.5% of zeolite powder, 6.98%-8.98% of microcrystalline cellulose, 0%-2% of sodium silicate, 10.19% of flour, 15% of cassava starch, 2.04% of fish oil, 2.04% of soybean oil, 0.5% of choline chloride, 1.75% of monocalcium phosphate, 0.4% of vitamin premix, 0.6% of trace element premix, 0.05% of allicin, 0.12% of lysine and 0.33% of methionine. By adding a proper amount of sodium silicate into the feed, the carps under heavy metal cadmium stress can be protected, the intestinal oxidative stress of the carps under the heavy metal cadmium stress is relieved, and the immune barrier is enhanced.

Description

Technical Field

[0001] This invention relates to the field of aquatic feed technology, and more specifically, to a feed that enhances the ability of juvenile carp to cope with heavy metal cadmium stress. Background Technology

[0002] Cadmium (Cd) stress has negative effects on aquatic animals, impacting the quality and safety of aquatic products and the profitability of aquaculture.

[0003] In related technologies, common methods for alleviating or eliminating Cd stress in aquaculture include improving aquaculture conditions, standardizing aquaculture management, adjusting feeding strategies, adding nutrients, and selecting superior breeds. Among these, adding nutrients is one of the most common and effective methods to alleviate the negative effects of Cd stress, as it can increase the body's energy supply and alleviate or eliminate the negative effects caused by Cd stress.

[0004] Current research on nutrients that alleviate Cd stress mainly focuses on trace elements, polysaccharides, probiotics, phenolic substances, vitamins, and sulfur-containing amino acids, but their ability to alleviate or prevent Cd stress has a low effect on the health of carp.

[0005] It should be noted that the information disclosed in the background section above is only used to enhance the understanding of the background of this disclosure, and therefore may include information that does not constitute prior art known to those skilled in the art. Summary of the Invention

[0006] The technical objective of this application is to address the above-mentioned shortcomings by providing a feed that enhances the ability of juvenile carp to cope with heavy metal cadmium stress. This application, by adding an appropriate amount of sodium silicate to the feed, can protect carp under heavy metal cadmium stress, reduce intestinal oxidative stress in carp under heavy metal cadmium stress, and enhance the immune barrier.

[0007] To achieve the above objectives, this application provides the following technical solution: According to one aspect of this application, a feed for enhancing the ability of juvenile carp to cope with heavy metal cadmium stress is provided, the feed comprising the following raw materials by weight percentage: fish meal 8%, chicken meal 5%, soybean meal 30%, chlorella 4%, cottonseed protein concentrate 6.5%, zeolite powder 4.5%, microcrystalline cellulose 6.98%–8.98%, sodium silicate 0%–2%, wheat flour 10.19%, cassava starch 15%, fish oil 2.04%, soybean oil 2.04%, choline chloride 0.5%, calcium dihydrogen phosphate 1.75%, vitamin premix 0.4%, trace element premix 0.6%, allicin 0.05%, lysine 0.12%, and methionine 0.33%.

[0008] In some embodiments, the feed is composed of the following ingredients by weight percentage: fish meal 8%, chicken meal 5%, soybean meal 30%, chlorella 4%, cottonseed protein concentrate 6.5%, zeolite powder 4.5%, microcrystalline cellulose 8.48%, sodium silicate 0.5%, wheat flour 10.19%, cassava starch 15%, fish oil 2.04%, soybean oil 2.04%, choline chloride 0.5%, calcium dihydrogen phosphate 1.75%, vitamin premix 0.4%, trace element premix 0.6%, allicin 0.05%, lysine 0.12%, and methionine 0.33%.

[0009] In some embodiments, each kilogram of the vitamin premix contains 8000 IU of VA, 500 mg of VC, 3000 IU of VD3, 60 mg of VE, 5 mg of VK3, 30 mg of VB2, 15 mg of VB6, 0.5 mg of VB12, 5000 mg of choline chloride, 175 mg of niacin, 2.5 mg of D-biotin, 1000 mg of inositol, 5 mg of folic acid, and 50 mg of pantothenic acid.

[0010] In some embodiments, each kilogram of the trace element premix contains 25 mg of zinc, 3 mg of copper, 25 mg of iron, 15 mg of manganese, 0.6 mg of iodine, 0.1 mg of cobalt, and 0.4 mg of selenium.

[0011] In some embodiments, the feed has a crude protein content of 29% and a crude fat content of 6%.

[0012] In some embodiments, the carp is Matsuura Mirror Carp.

[0013] In some embodiments, the initial weight of the Matsuura mirror carp is 6.60 ± 0.04 g.

[0014] According to another aspect of this application, a method for preparing feed to enhance the ability of juvenile carp to cope with heavy metal cadmium stress is provided, comprising: crushing the raw materials and mixing them evenly according to the formula, making them into 2 mm pellet feed using a three-phase asynchronous motor, and storing them in a -20°C refrigerator.

[0015] According to another aspect of this application, the use of the feed described herein or the feed prepared by the described method in feeding juvenile carp is provided.

[0016] In some embodiments, the carp is a Matsuura mirror carp, and the juvenile fish weighs 6.60 ± 0.04 g.

[0017] Compared with the prior art, the advantages and positive effects of this application are as follows: by adding an appropriate amount of sodium silicate to the feed, this application can protect carp under heavy metal cadmium stress, reduce intestinal oxidative stress of carp under heavy metal cadmium stress, and enhance the immune barrier. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 This paper illustrates the effect of sodium silicate in the feed of the present application embodiments on the serum biochemistry of Matsuura mirror carp under waterborne cadmium stress.

[0020] Figure 2 This paper illustrates the effect of sodium silicate in the feed of the present application on the intestinal antioxidant enzymes of Matsuura mirror carp under waterborne cadmium stress.

[0021] Figure 3 This paper illustrates the effect of sodium silicate in the feed of the present application on the intestinal antioxidant genes of Matsuura mirror carp under waterborne cadmium stress.

[0022] Figure 4 This paper illustrates the effect of sodium silicate in the feed of the present application embodiments on the intestinal tight junction factor of Matsuura mirror carp under waterborne cadmium stress.

[0023] Figure 5 This paper illustrates the effect of sodium silicate in the feed of the present application on pro-inflammatory factors in the intestine of Matsuura mirror carp under waterborne cadmium stress. Detailed Implementation

[0024] To better understand the above-mentioned objectives, features, and advantages of this application, the application will be further described below with reference to the accompanying drawings and embodiments. It should be noted that, unless otherwise specified, the embodiments and features described in the embodiments of this application can be combined with each other.

[0025] The present application will be further described below with reference to the accompanying drawings and specific embodiments.

[0026] 1. Experimental Method: The experiment used fish meal, chicken meal, soybean meal, Chlorella and cottonseed protein concentrate as the main protein sources, flour and tapioca starch as sugar sources, and fish oil and soybean oil as fat sources to make compound feed.

[0027] Sodium silicate was added to the experimental feed at concentrations of 0%, 0.5%, and 2% (Table 1). The feed ingredients were crushed, mixed evenly according to the formula, and made into 2 mm pellets using a three-phase asynchronous motor (Y2-112M-4, Weihai Taifu Xima Motor Co., Ltd.). The pellets were then stored in a -20℃ refrigerator.

[0028] The fish used in the experiment was the Matsuura mirror carp ( Cyprinus carpio Songpu, a fish species endemic to China, is known for its delicate and thick flesh, rapid growth, high survival rate, and large-scale aquaculture. Stress experiments were conducted in the fish nutrition and feed aquaculture workshop of the Heilongjiang Fisheries Research Institute. Acute stress was introduced with Cd after 56 days of rearing. Feeding was stopped for 1 day before the experiment began. Fifteen Songpu mirror carp (6.60±0.04 g each) were selected from groups with sodium silicate additions of 0%, 0.5%, and 2%. Each aquarium was filled with 1.25 mg / L of Cd, and the same amount of Cd was changed daily. 2+ The water concentration was adjusted, with 1 / 3 of the total volume replaced daily, and the stress period was 96 h. The experimental water was at (25±1)℃, dissolved oxygen concentration >6.0 mg / L, and pH (7.8±0.2). The water was changed regularly and feces were removed promptly. The experiment lasted for 96 h, with sampling times at 0, 12, 24, 48, and 96 h.

[0029] Table 1 Feed formulation and nutrient levels (dry matter basis)

[0030] Note: The following vitamin premix is ​​provided per kilogram of diet: VA 8000 IU, VC 500 mg, VD3 3000 IU, VE 60 mg, VK3 5 mg, VB2 30 mg, VB6 15 mg, VB12 0.5 mg, choline chloride 5000 mg, niacin 175 mg, D-biotin 2.5 mg, inositol 1000 mg, folic acid 5 mg, pantothenic acid 50 mg. The following micronutrient premix is ​​provided per kilogram of diet: zinc 25 mg, copper 3 mg, iron 25 mg, manganese 15 mg, iodine 0.6 mg, cobalt 0.1 mg, selenium 0.4 mg. Nutrient levels are measured values.

[0031] 2. Sample collection: Sampling time points were 0, 12, 24, 48, and 96 hours. At each time point, blood was drawn from 3 fish under anesthesia with 100 mg / L MS-222. The intestine of one fish was separated and stored at -20°C for the determination of antioxidant enzymes. The intestine of one fish was stored in 4% paraformaldehyde for the determination of intestinal morphology. The intestine of one fish was sealed in aluminum foil, placed in liquid nitrogen, and stored at -80°C for gene detection.

[0032] 3. Serum biochemistry and antioxidant enzyme assays: After centrifugation, serum samples were thawed at 4°C and serum biochemical assays were performed. Kits for total protein (TP), albumin (ALB), total cholesterol (T-CHO), and triglycerides (TG) were purchased from Nanjing Jiancheng Bioengineering Institute. A glucose (GLU) kit was purchased from Shanghai Rongsheng Biopharmaceutical Co., Ltd.

[0033] The intestinal contents to be tested were collected on ice and added to 0.9% pre-cooled physiological saline at a mass (g): volume (ml) ratio of 1:9. The mixture was homogenized into a 10% homogenate using a high-speed dispersion homogenizer (FJ-200). After centrifugation, the supernatant was aliquoted and stored at -20℃ for the determination of various enzyme activities. Catalase (CAT), glutathione peroxidase (GPX), glutathione reductase (GR), and malondialdehyde (MDA) were measured using standard kits from Nanjing Jiancheng Biological Research Institute. Superoxide dismutase (SOD) was purchased from Beijing Baoruyi Biotechnology Co., Ltd.

[0034] 4. Gut gene expression levels: Take a pre-cooled mortar (-80℃), add an appropriate amount of liquid nitrogen, and grind the intestine into powder. Aliquot the powder into pre-cooled, enzyme-free EP tubes. Extract total RNA from the intestine using RNAiso reagent, and detect the RNA concentration using a spectrophotometer. Prepare the reaction solution for quantitative real-time reverse transcription according to the TaKaRa kit instructions. Reverse transcribe the RNA into cDNA, store at -80℃, and use for quantitative real-time detection.

[0035] The volume of quantitative real-time PCR (qPCR) was 10 μL, using Takara TB Green Premix Ex. Taq II (Tli RNaseH Plus), the fluorescence quantitative reagent was prepared according to the instructions. Each sample was tested three times. Measurements were performed using a real-time PCR system (Applied Biosystems 7500) with a threshold cycling (2... -ΔΔCt The relative expression levels of the gene were calculated using the following method. Primers were designed using NCBI and Primer 5.0 software, with β-actin as the internal reference gene.

[0036] 5. Statistical Analysis: Experimental data were subjected to multiple comparisons (Duncan's) and one-way ANOVA, and the results are presented in a bar chart. P A value <0.05 indicates a statistically significant difference.

[0037] 6. Experimental Results: (1) Effects on serum biochemistry: Figure 1 This paper illustrates the effect of sodium silicate in the feed of an embodiment of this application on the serum biochemistry of Matsuura mirror carp under waterborne cadmium stress. Figure 1 It was found that when the sodium silicate addition was 0%, the ALB content reached its highest value at 0 h, significantly higher than at 96 h (P<0.05); the serum LDL-C and HDL-C contents reached their highest values ​​at 48 h, significantly higher than at other time points (P<0.05). When the sodium silicate addition was 0.5%, the ALB, T-CHO, LDL-C, and HDL-C contents reached their highest values ​​at 24 h, with T-CHO and LDL-C contents significantly higher than at 0 h (P<0.05). When the sodium silicate addition was 2%, the ALB content reached its highest value at 12 h, but there was no significant difference compared to 24 and 48 h (P>0.05); the serum T-CHO, LDL-C, and HDL-C contents reached their highest values ​​at 48 h.

[0038] (2) Effects on intestinal antioxidant enzymes: Figure 2 This paper illustrates the effect of sodium silicate in the feed of an embodiment of this application on the intestinal antioxidant enzymes of Matsuura mirror carp under waterborne cadmium stress. Figure 2 It was found that when the sodium silicate addition was 0%, the activities of SOD, CAT, and GR in the intestine reached their highest values ​​at 24 h; at 48 h, the GSH activity reached its highest value, and the MDA content reached its lowest value. When the sodium silicate addition was 0.5%, the activities of SOD, CAT, and GSH in the intestine reached their highest values ​​at 12 h; at 24 h, the GR activity reached its highest value, and the MDA activity reached its lowest value. When the sodium silicate addition was 2%, the activities of SOD, GR, and GSH in the intestine reached their highest values ​​at 24 h, significantly higher than at 0 h (P<0.05); the CAT activity reached its highest value at 12 h, and the MDA content reached its lowest value at 24 h.

[0039] (3) Effects on intestinal antioxidant genes Figure 3 This paper illustrates the effect of sodium silicate in the feed of embodiments of this application on the intestinal antioxidant genes of Matsuura mirror carp under waterborne cadmium stress. Figure 3It was found that when the sodium silicate addition was 0%, the expression levels of Keap1 and CuMnSOD in the intestine reached their highest values ​​at 96 h; the expression levels of Nrf2 and CAT reached their highest values ​​at 0 h, significantly higher than at other time points (P<0.05); and the expression level of GPX1a reached its highest value at 24 h, significantly higher than at 0 h (P<0.05). When the sodium silicate addition was 0.5%, the expression levels of Keap1, CuMnSOD, and GPX1a all reached their highest values ​​at 12 h, significantly higher than at 0 h (P<0.05); and the expression levels of Nrf2 and CAT reached their highest values ​​at 24 h. When the sodium silicate addition was 2%, the expression levels of Keap1, Nrf2, and CuMnSOD reached their highest values ​​at 24 h; and the expression levels of CAT and GPX1a reached their highest values ​​at 12 h, significantly higher than at 0 h (P<0.05).

[0040] (4) Effects on gut tight junction factors Figure 4 This paper illustrates the effect of sodium silicate in the feed of embodiments of this application on the intestinal tight junction factor of Matsuura mirror carp under waterborne cadmium stress. Figure 4 The results showed that when the sodium silicate addition was 0%, the expression levels of occludin and ZO-1 in the intestine reached their highest values ​​at 96 h, significantly higher than at other time points (P<0.05). With increasing stress time, MLCK expression decreased; the expression levels of claudin3 and claudin7 reached their highest values ​​at 48 h, significantly higher than at 0 h (P<0.05). When the sodium silicate addition was 0.5%, the expression levels of occludin and claudin7 reached their highest values ​​at 24 h, significantly higher than at other time points (P<0.05); the expression levels of ZO-1, MLCK, and claudin3 reached their highest values ​​at 12 h. When the sodium silicate addition was 2%, the expression levels of occludin, MLCK, claudin3, and claudin7 reached their highest values ​​at 24 h, significantly higher than at other time points (P<0.05); the expression level of ZO-1 reached its highest value at 12 h.

[0041] (5) Effects on intestinal pro-inflammatory factors Figure 5 This paper illustrates the effect of sodium silicate in the feed of an embodiment of this application on pro-inflammatory factors in the intestine of Matsuura mirror carp under waterborne cadmium stress. Figure 5It was found that when the sodium silicate addition was 0%, the expression levels of interleukin-1β (IL-β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNFα) in the intestine reached their highest values, significantly higher than those in the other groups (P<0.05); the expression level of interleukin-10 (IL-10) reached its highest value at 48 h, with no significant difference compared to 24 h (P>0.05). When the sodium silicate addition was 0.5%, the expression levels of IL-β and IL-10 reached their highest values ​​at 12 h, and the expression levels of IL-6 and TNFα reached their highest values ​​at 24 h, significantly higher than those at 0 h (P<0.05). When the sodium silicate addition was 2%, the expression levels of IL-β, IL-6, and IL-10 reached their highest values ​​at 24 h, and the expression level of TNFα reached its highest value at 12 h.

[0042] The specific names of the above-mentioned indicators are: Kelch-like ECH-associated protein 1; nuclear factor-erythroid 2-realized factor 2 (Nrf2); Copper-manganese superoxide dismutase (CuMnSOD); catalase (CAT); glutathione peroxidase 1a (GPX1a); glutathione peroxidase 1b (GPX1b); interleukin-1β (IL-1β); interleukin-6 (IL-6); interleukin-8 (IL-8); interleukin-10 (IL-10); tumor necrosis factor α (TNF-α); occludin; and zonal occluden protein. 1, ZO-1; Claudin3 (closing protein 3); Claudin7 (closing protein 7); Claudin11 (closing protein 11).

[0043] In conclusion, sodium silicate can alleviate intestinal oxidative stress in carp under Cd stress and enhance the immune barrier. Carp feed containing 0.5% sodium silicate can be considered a novel functional feed to enhance carp's resistance to heavy metal cadmium stress, and can be used to prevent aquaculture and food safety problems caused by Cd stress.

[0044] Through the above specific embodiments, those skilled in the art can easily implement this application. However, it should be understood that this application is not limited to the specific embodiments described above. Based on the disclosed embodiments, those skilled in the art can arbitrarily combine different technical features to achieve different technical solutions.

Claims

1. A feed for enhancing the ability of juvenile carp to cope with heavy metal cadmium stress, characterized in that, The feed is composed of the following ingredients by weight percentage: fish meal 8%, chicken meal 5%, soybean meal 30%, chlorella 4%, cottonseed protein concentrate 6.5%, zeolite powder 4.5%, microcrystalline cellulose 6.98%–8.98%, sodium silicate 0%–2%, wheat flour 10.19%, cassava starch 15%, fish oil 2.04%, soybean oil 2.04%, choline chloride 0.5%, calcium dihydrogen phosphate 1.75%, vitamin premix 0.4%, trace element premix 0.6%, allicin 0.05%, lysine 0.12%, and methionine 0.33%.

2. The feed for enhancing the ability of juvenile carp to cope with heavy metal cadmium stress according to claim 1, characterized in that, The feed is composed of the following ingredients by weight percentage: fish meal 8%, chicken meal 5%, soybean meal 30%, chlorella 4%, cottonseed protein concentrate 6.5%, zeolite powder 4.5%, microcrystalline cellulose 8.48%, sodium silicate 0.5%, wheat flour 10.19%, cassava starch 15%, fish oil 2.04%, soybean oil 2.04%, choline chloride 0.5%, calcium dihydrogen phosphate 1.75%, vitamin premix 0.4%, trace element premix 0.6%, allicin 0.05%, lysine 0.12%, and methionine 0.33%.

3. The feed for enhancing the ability of juvenile carp to cope with heavy metal cadmium stress according to claim 1, characterized in that, Each kilogram of the vitamin premix contains 8000 IU of VA, 500 mg of VC, 3000 IU of VD3, 60 mg of VE, 35 mg of VK, 30 mg of VB2, 15 mg of VB6, 0.5 mg of VB12, 5000 mg of choline chloride, 175 mg of niacin, 2.5 mg of D-biotin, 1000 mg of inositol, 5 mg of folic acid, and 50 mg of pantothenic acid.

4. The feed for enhancing the ability of juvenile carp to cope with heavy metal cadmium stress according to claim 1, characterized in that, Each kilogram of the aforementioned trace element premix contains 25 mg of zinc, 3 mg of copper, 25 mg of iron, 15 mg of manganese, 0.6 mg of iodine, 0.1 mg of cobalt, and 0.4 mg of selenium.

5. The feed for enhancing the ability of juvenile carp to cope with heavy metal cadmium stress according to claim 1, characterized in that, The feed has a crude protein content of 29% and a crude fat content of 6%.

6. The feed for enhancing the ability of juvenile carp to cope with heavy metal cadmium stress according to claim 1, characterized in that, The carp in question is the Matsuura Mirror Carp.

7. The feed for enhancing the ability of juvenile carp to cope with heavy metal cadmium stress according to claim 6, characterized in that, The initial weight of the Matsuura mirror carp was 6.60 ± 0.04 g.

8. A method for preparing a feed to enhance the ability of juvenile carp to cope with heavy metal cadmium stress, as described in any one of claims 1-7, characterized in that, include: The raw materials are crushed and mixed evenly according to the formula, and then made into 2 mm pellet feed using a three-phase asynchronous motor and stored in a -20℃ refrigerator.

9. The use of the feed according to any one of claims 1-7 or the feed prepared by the preparation method according to claim 8 in feeding juvenile carp.

10. The application according to claim 9, characterized in that, The carp in question is a Matsuura mirror carp, and the weight of the juvenile fish is 6.60±0.04g.

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