Feed formula for improving growth, digestion and molting of procambarus clarkii by adding vitamin E

Abstract

The invention provides a feed formula for improving growth, digestion and molting of procambarus clarkii by adding vitamin E. Based on the feed formula meeting the nutritional requirements of the procambarus clarkii, by adding the vitamin E (0-0.1%), the growth performance of adult procambarus clarkii can be remarkably improved after the feed formula is used for 56 days, and periodic molting is effectively promoted. Vitamin E promotes ecdysis of the procambarus clarkia by regulating periodic ecdysis behaviors, so that the growth performance of the procambarus clarkia is improved, and the procambarus clarkia is of great significance in promoting development of the procambarus clarkia breeding industry.

Description

Technical Field

[0001] The present invention belongs to the technical field of shrimp feed, and specifically relates to a feed formulation method for improving the growth, digestion and molting of Procambarus clarkii. Background Art

[0002] Procambarus clarkii, commonly known as crayfish, belongs to the phylum Arthropoda, subphylum Crustacea, and order Decapoda. Procambarus clarkii has a short growth cycle, strong reproductive ability, and extremely strong environmental adaptability. After years of cultivation and promotion, Procambarus clarkii has now become an important part of China's aquaculture industry and has become a kind of aquatic product with both high economic benefits and rich nutritional value. In 2023, the aquaculture area of Procambarus clarkii in China reached 29.5 million mu, the aquaculture output reached 3.161 million tons, and the total comprehensive output value of the aquaculture industry reached 467.6 billion yuan. With the continuous expansion and innovation of the aquaculture scale and mode, germplasm degradation has become one of the main reasons restricting the sustainable development of the Procambarus clarkii aquaculture industry. The reasons affecting germplasm degradation include unbalanced feed nutrition, excessive stocking density, long-term inbreeding, insufficient supply of high-quality seedlings, etc. Moreover, in the pond aquaculture mode, shrimp diseases occur frequently during aquaculture, and the prevention and control is difficult. When feeding Procambarus clarkii with artificial compound feed, problems such as incomplete molting and slow growth are likely to occur during the aquaculture period, which greatly reduces the economic benefits of aquaculture, causes serious economic losses to the aquaculture industry, and is not conducive to the healthy and sustainable development of the industry. Therefore, developing green, environmentally friendly, healthy and safe feed has become the top priority of the aquaculture industry.

[0003] In view of this, the present invention is specifically proposed. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to overcome the deficiencies of the prior art and provide a feed formulation for adding vitamin E to improve the growth, digestion and molting of Procambarus clarkii, so as to solve the problems of incomplete molting and slow growth of Procambarus clarkii.

[0005] To achieve the above purpose, the present invention adopts the following technical scheme: A feed formulation for adding vitamin E to improve the growth, digestion and molting of Procambarus clarkii, the components and contents thereof include:

[0006] Vitamin E 0 - 0.1%, fish meal 5%, soybean meal 15%, cottonseed protein 6%, rapeseed meal 13%, peanut meal 10%, corn distillers grains 5%, wheat flour 28%, sodium alginate 2%, calcium dihydrogen phosphate 2.3%, spray-dried yeast powder 2%, soybean oil 3.95%, soy lecithin 1.5%, cholesterol 0.5%, vitamin premix 1%, inorganic salt premix 1%, sodium chloride 0.2%, choline chloride 0.25%, vitamin C 0.25%, chitosan 0.1%, L-threonine 0.35, L-lysine 0.3%, methionine 0.1%, cellulose 2.1 - 2.2%.

[0007] All percentages are by weight.

[0008] Among them, based on each kilogram of basal diet, the addition amount of vitamin E is 0 - 1000 mg;

[0009] Preferably, based on each kilogram of basal diet, the addition amount of vitamin E is 50 - 400 mg;

[0010] More preferably, based on each kilogram of basal diet, the addition amount of vitamin A is 50 - 200 mg.

[0011] Further preferably, based on each kilogram of basal diet, the addition amount of vitamin A is 100 - 200 mg.

[0012] Furthermore, the vitamin premix and its content include:

[0013] Vitamin A: 4 g / kg, Vitamin B1: 10 g / kg, Vitamin B2: 10 g / kg, Vitamin B6: 20 g / kg, Vitamin B12: 0.01 g / kg, Vitamin C: 20 g / kg, Vitamin D: 0.02 g / kg, Vitamin K3: 10 g / kg, Niacin: 40 g / kg, Biotin: 0.2 g / kg, Calcium Pantothenate: 20 g / kg, Folic Acid: 0.5 g / kg, Inositol: 400 g / kg.

[0014] Furthermore, the components and content of the inorganic salt premix include:

[0015] Potassium Iodate: 0.6 g / kg, Sodium Selenite Pentahydrate: 0.08 g / kg, Potassium Dihydrogen Phosphate: 320 g / kg, Magnesium Sulfate: 200 g / kg, Manganese Sulfate Monohydrate: 20 g / kg, Copper Chloride Dihydrate: 2 g / kg, Zinc Sulfate Heptahydrate: 60 g / kg, Ferrous Sulfate Heptahydrate: 50 g / kg, Sodium Chloride: 100 g / kg, Cobalt Chloride Hexahydrate: 2 g / kg. Description of the Drawings

[0016] Figure 1: Optimal addition amount of vitamin E in Procambarus clarkii feed; E: E cell (embryonic cell), F: F cell (fibrocyte), and C: stellate lumen. Magnification 100× (100 μm).

[0017] Figure 2 : Effect of dietary vitamin E level on intestinal tissue morphology of Procambarus clarkii; (A) CL: chitin layer; EL: epithelial layer; CT: connective tissue layer; M: muscle bundle; ML: muscle layer; Magnification 100× (100 μm). (B) Effect of dietary vitamin E level on intestinal villus length and width of Procambarus clarkii.

[0018] Figure 3 : Effect of dietary vitamin E level on molting regulatory factors of Procambarus clarkii. MIH, molt-inhibiting hormone; mTOR, mammalian target of rapamycin; S6K1, ribosomal protein S6K kinase 1; 4EBP1, 4E-binding protein 1; EcR, ecdysone receptor; RXR, retinoid X receptor; E75; Chitinase, chitinase. Detailed implementation manners

[0019] The following will elaborate on the implementation manners of the present invention with the aid of specific embodiments. However, those skilled in the art should clearly understand that the listed embodiments are only intended to illustrate the present invention and do not constitute a limitation on the scope of the present invention. For the specific conditions not clearly mentioned in the embodiments, they should all be understood as being carried out according to industry conventions or the conditions recommended by manufacturers. In addition, for the reagents or instruments involved, if the manufacturer is not specifically indicated, they all refer to commercially available conventional products and are easily obtainable.

[0020] Vitamin E is a fat-soluble vitamin that can be hydrolyzed to produce tocopherol. It is soluble in organic solvents such as fats and ethanol, is sensitive to oxygen, stable to acids and heat, and unstable to alkalis. Since it can only be synthesized by photosynthetic organisms, it must be supplemented in the feed of aquatic animals. A large number of studies have shown that vitamin E has the functions of promoting the growth, reproduction, immunity, feeding of aquatic animals, and protecting essential fatty acids from oxidation. Vitamin E can also act synergistically with propionamide, vitamin C, selenium-containing compounds, astaxanthin, arachidonic acid, etc. to improve the growth, antioxidant and other properties of the body. Therefore, vitamin E plays an important role in the healthy growth and development of Procambarus clarkii.

[0021] The following introduces a specific feed formula for adding vitamin E to improve the growth, digestion and molting of Procambarus clarkii provided in the embodiments of the present application.

[0022] The feed formula method for improving the growth, digestion and molting of Procambarus clarkii provided in the embodiments of the present application, the components and contents of the compound feed include:

[0023] Vitamin E 0 - 0.1%, fish meal 5%, soybean meal 15%, cottonseed protein 6%, rapeseed meal 13%, peanut meal 10%, corn distillers grains 5%, wheat flour 28%, sodium alginate 2%, calcium dihydrogen phosphate 2.3%, spray-dried yeast powder 2%, soybean oil 3.95%, soy lecithin 1.5%, cholesterol 0.5%, vitamin premix 1%, inorganic salt premix 1%, sodium chloride 0.2%, choline chloride 0.25%, vitamin C 0.25%, chitosan 0.1%, L-threonine 0.35, L-lysine 0.3%, methionine 0.1%, cellulose 2.1 - 2.2%. All percentages are by weight.

[0024] As a specific example, according to the feed formula in Table 1 below, six isonitrogenous and isolipidic feeds were formulated, named E0, E1, E2, E3, E4, and E5 respectively. Among them, the feed without added vitamin E was used as the control group, and vitamin E was added to the same feed as the control group at the addition levels of 0.005%, 0.01%, 0.02%, 0.04%, and 0.1%. The above feeds were used as experimental feeds for the breeding experiment.

[0025] Table 1 Experimental feed formula and nutritional composition

[0026]

[0027] The subscripts in Table 1 are annotated below;

[0028] a : Each kilogram of vitamin premix includes: vitamin A 4 g, vitamin D 0.02 g, vitamin K3 10 g, vitamin B1 10 g, vitamin B2 10 g, vitamin B6 20 g, niacin 40 g, biotin 0.2 g, calcium pantothenate 20 g, folic acid 0.5 g, vitamin B12 0.01 g, vitamin C 20 g, inositol 400 g;

[0029] b : Each kilogram of mineral premix includes: potassium iodate 0.6 g, sodium selenite pentahydrate 0.08 g, potassium dihydrogen phosphate 320 g, magnesium sulfate 200 g, manganese sulfate monohydrate 20 g, copper chloride dihydrate 2 g, zinc sulfate heptahydrate 60 g, ferrous sulfate heptahydrate 50 g, sodium chloride 100 g, cobalt chloride hexahydrate 2 g.

[0030] Specific experimental process

[0031] a. Feeding process

[0032] The breeding experiment was carried out in plastic round barrels (diameter × height = 190 cm × 70 cm). Before the formal breeding, Procambarus clarkii were temporarily cultured in plastic round barrels to adapt to the environment. After two weeks of temporary culture, the formal breeding experiment began. The experiment used 720 healthy Procambarus clarkii (initial weight: 8.14 ± 0.05 g). After fasting for 24 hours, they were randomly assigned to 24 breeding barrels, with 30 shrimps in each barrel. A total of 6 groups were divided, with 4 replicates in each group. During the breeding period, they were fed twice a day (8:00 and 16:00) to satiety, and the feeding rate was 3%-5% of the body weight. The breeding experiment lasted for 8 weeks, the water temperature was 24-28 °C, the pH of the breeding water was 8.0-8.5, the dissolved oxygen > 5 mg / L, and the ammonia nitrogen < 0.05 mg / L.

[0033] b. Sample collection

[0034] At the end of the breeding experiment, after Procambarus clarkii were starved for 24 hours, all the shrimps were fished out, the surface moisture was blotted dry, weighed, and the number of surviving tails in each experimental barrel was counted for calculating the growth index. The hepatopancreas and intestine were quickly taken out on ice, and part of the hepatopancreas and intestine were separated and placed in 4% paraformaldehyde for preparing hematoxylin-eosin (H.E) staining of the hepatopancreas and intestine and oil red O staining sections of the hepatopancreas; randomly collect the hepatopancreas and intestine of 5 shrimps in each barrel, quickly transfer them to liquid nitrogen, and store them in a -80 °C refrigerator for subsequent real-time fluorescence quantitative PCR analysis.

[0035] c. Sample analysis (i) Tissue morphology observation

[0036] After the intestinal tissue specimens were fixed, they were dehydrated, cleared, paraffin-embedded, serially sectioned (thickness: 4 μm), and then stained with H.E and oil red O. An optical microscope (SOPTOPEX31, Shunyu Optoelectronic Technology (Group) Co., Ltd.) was used to observe and photograph the stained sections, and an image analysis system (Image Pro Plus 6.0) was used to measure and count the length and width of the intestinal villi.

[0037] (ii) Real-time fluorescence detection

[0038] After the test samples were ground and broken, total RNA was extracted using the Total RNAkitI kit (OmegaBio-Tek, USA). After detecting its integrity by 1% agarose gel electrophoresis, a ultra-micro spectrophotometer was used to measure the concentration and purity of the total RNA. Use MonScript TMThe RTIII Super Mix with dsDNase (Two-Step) kit (Monad Biotech Co., Ltd., China) was used to reverse transcribe the extracted total RNA into cDNA. Real-time quantitative PCR was performed using the Line Gene 9600 Plus real-time PCR system (Bioer Technology Co., Ltd., China). The 20 μL reaction system consisted of: 0.4 μL of forward and reverse primers (10 μmol / L), 1.5 μL of cDNA, 10 μL of MonAmp TM ChemoHS qPCR mix (Monad Biotech Co., Ltd., China) and 7.7 μL of nuclease-free water. The real-time quantitative PCR program was as follows: 95 °C for 10 min, 95 °C for 10 s, 60 °C for 10 s, 72 °C for 30 s, for a total of 40 cycles. Using β-actin as the internal reference gene, the relative expression level of mRNA was calculated using the 2 -△△CT -method.

[0039] d. Data analysis

[0040] Formulas for calculating growth performance evaluation indicators

[0041] Survival rate (SR, %) = number of survivors / total number × 100;

[0042] Weight gain rate (WGR, %) = (final weight - initial weight) / initial weight × 100;

[0043] Specific growth rate (SGR, % / d) = (ln final weight - ln initial weight) / number of culture days × 100;

[0044] Feed conversion ratio (FCR) = feed consumption / weight gain;

[0045] Hepatossomatic index (HSI, %) = (weight of digestive gland / body weight) × 100; The experimental data were analyzed by one-way ANOVA using SPSS 20.0 software (IBM, USA), and multiple comparisons were performed using the Tukey method. The significance level was P < 0.05, and the analysis results were expressed as mean ± standard deviation The way of. The experimental data of weight gain rate and feed conversion ratio were fitted with a broken line model using Origin 2019 software (OriginLab Company, USA) to obtain the optimal vitamin E requirement for Procambarus clarkii feed.

[0046] e. Experimental results (i) Growth performance

[0047] After feeding with feeds at different vitamin addition levels, the growth performance results of Procambarus clarkii in each group are shown in Table 2 below. There was no significant difference in survival rate among groups (P>0.05). With the increase of vitamin E addition amount, the final average weight, weight gain rate and specific growth rate of Procambarus clarkii all increased first and then decreased, reaching the highest in groups E2 and E3 (P<0.05). The feed coefficient of group E3 was significantly lower than that of group E5 (P<0.05). Adding 100 and 200 mg / kg of vitamin E to the feed significantly reduced the hepatosomatic index of Procambarus clarkii (P<0.05).

[0048] Table 2 Effects of vitamin E on the growth performance of Procambarus clarkii

[0049]

[0050] (ii) Activities of digestive enzymes in hepatopancreas and intestine

[0051] The experimental results in Table 3 showed that there was no significant difference in the activities of trypsin in the hepatopancreas and intestine of Procambarus clarkii among groups (P>0.05), but after adding vitamin E, the activities of amylase in the hepatopancreas and lipase in the intestine were significantly higher than those of the control group (P<0.05). The activity of lipase in the hepatopancreas increased with the increase of vitamin E addition level. When the addition level was 100-1000 mg / kg, the lipase activity was significantly higher than that of the control group (P<0.05).

[0052] Table 3 Effects of vitamin E on the activities of digestive enzymes in the hepatopancreas and intestine of Procambarus clarkii

[0053]

[0054] (iii) Histological observation

[0055] The intestinal tissue morphology of Procambarus clarkii is as Figure 2 shown. Compared with the control group, when the vitamin E level was 100 mg / kg and 200 mg / kg, the intestinal villi of Procambarus clarkii had regular shapes, were arranged more closely, the small folds on their surfaces were more developed, the compactness of connective tissue was better, and the muscle bundles wrapped were more complete. With the increase of vitamin A level in the feed, the intestinal villi length showed a trend of increasing first and then decreasing, and reached the maximum value when the vitamin A level in the feed was 200 mg / kg (P<0.05).

[0056] (iv) Molting regulatory factors of Procambarus clarkii

[0057] The effects of vitamin E level in the feed on the molting regulatory factors of Procambarus clarkii are as Figure 3As shown in the figure. Compared with the control group, when the vitamin E level was 200 mg / kg, the relative expression levels of mTOR, S6K1, E75, EcR, RXR, and Chitinase genes in the hepatopancreas were significantly up-regulated, and the relative expressions of 4EBP1 and MIH were significantly down-regulated (P<0.05).

[0058] In summary, in this experiment, vitamin E (200 mg / kg) significantly up-regulated the relative expression level of the chitinase gene in the hepatopancreas compared with the control group, accelerated the synthesis of the exoskeleton of Procambarus clarkii, and promoted its periodic molting. The above results indicate that vitamin E promotes the molting of Procambarus clarkii by regulating the periodic molting behavior (molting occurrence and new shell synthesis), thereby improving the growth performance of Procambarus clarkii.

[0059] As described above, only the preferred specific embodiments of the present application are given, but the protection scope of the present application is not limited thereto. Any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present application should be covered by 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. A feed formula for improving the growth, digestion and molting of Procambarus clarkii by adding vitamin E, characterized in that, The components and contents of the formulated feed include: Vitamin E 0 - 0.1%, fish meal 5%, soybean meal 15%, cottonseed protein 6%, rapeseed meal 13%, peanut meal 10%, corn distillers grains 5%, wheat flour 28%, sodium alginate 2%, calcium dihydrogen phosphate 2.3%, spray-dried yeast powder 2%, soybean oil 3.95%, soy lecithin 1.5%, cholesterol 0.5%, vitamin premix 1%, inorganic salt premix 1%, sodium chloride 0.2%, choline chloride 0.25%, vitamin C 0.25%, chitosan 0.1%, L-threonine 0.35, L-lysine 0.3%, methionine 0.1%, cellulose 2.1 - 2.2% All percentages are by weight.

2. A feed formula for improving the growth, digestion and molting of Procambarus clarkii by adding vitamin E according to claim 1, characterized in that, The raw materials of the formulated feed include basic feed and vitamin E; Among them, based on each kilogram of basic feed, the addition amount of vitamin E is 0 - 1000 mg; Preferably, based on each kilogram of basic feed, the addition amount of vitamin E is 50 - 400 mg; More preferably, based on each kilogram of basic feed, the addition amount of vitamin A is 50 - 200 mg. Further preferably, based on each kilogram of basic feed, the addition amount of vitamin A is 100 - 200 mg.

3. The compound feed according to claim 1, wherein The plant protein includes: soybean meal, cottonseed protein, rapeseed meal, peanut meal, corn DDGS; And / or, the animal protein includes: fish meal; And / or, the flour includes: wheat flour; And / or, the vegetable oil includes: soybean oil, soy lecithin; And / or, the amino acids include: L-threonine, L-lysine, methionine.

4. The compound feed according to claim 1, characterized in that, The components and contents of the vitamin premix include: Vitamin A: 4 g / kg, Vitamin B1: 10 g / kg, Vitamin B2: 10 g / kg, Vitamin B6: 20 g / kg, Vitamin B12: 0.01 g / kg, Vitamin C: 20 g / kg, Vitamin D: 0.02 g / kg, Vitamin K3: 10 g / kg, Niacin: 40 g / kg, Biotin: 0.2 g / kg, Calcium Pantothenate: 20 g / kg, Folic Acid: 0.5 g / kg, Inositol: 400 g / kg.

5. The compound feed according to claim 1, characterized in that, The components and contents of the inorganic salt premix include: Potassium Iodate: 0.6 g / kg, Sodium Selenite Pentahydrate: 0.08 g / kg, Potassium Dihydrogen Phosphate: 320 g / kg, Magnesium Sulfate: 200 g / kg, Manganese Sulfate Monohydrate: 20 g / kg, Copper Chloride Dihydrate: 2 g / kg, Zinc Sulfate Heptahydrate: 60 g / kg, Ferrous Sulfate Heptahydrate: 50 g / kg, Sodium Chloride: 100 g / kg, Cobalt Chloride Hexahydrate: 2 g / kg.

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