Additive and feed for improving survival, growth and intestinal development of marine fish juveniles

Abstract

The invention belongs to the technical field of aquatic feed and feed additives, and particularly relates to an additive and feed for improving survival, growth and intestinal development of marine fish juveniles. The main component of the additive is melatonin, and the melatonin, microcrystalline cellulose and alpha-starch are reasonably proportioned, so that the intestinal development of the marine fish juveniles including the larimichthys crocea juveniles is effectively promoted, the digestion and absorption capacity is improved, the immunity is improved, and the survival and growth of the marine fish juveniles are facilitated.

Description

Technical Field

[0001] The present invention belongs to the technical field of aquatic feed and feed additives, and particularly relates to an additive and feed for improving the survival, growth and intestinal development of juvenile marine fish. Background Art

[0002] In recent years, with the rapid development of intensive mariculture, seedling cultivation has become a crucial link in its industrial chain. The intestine of fish, as an important digestive and immune organ, not only plays a key role in the digestion and absorption of nutrients, but also serves as the first line of defense against the invasion of pathogenic microorganisms. However, the juvenile stage is a critical period for the intestinal development of fish. At this time, the intestinal structure is not yet fully developed, the digestive enzyme activity is relatively low, and the immune barrier has not been completely established. Therefore, the juvenile intestine is extremely sensitive to external environmental stimuli and is easily damaged, leading to problems such as high mortality and high morbidity, which has become a key bottleneck restricting the sustainable development of seedling cultivation.

[0003] As an important marine cultured fish in China, the successful artificial breeding of large yellow croaker has laid a solid foundation for large-scale farming. However, the imperfect development of the digestive system in juveniles is still one of the important factors leading to its high mortality, seriously restricting the healthy and sustainable development of the large yellow croaker aquaculture industry. Therefore, the development of targeted functional additives and feeds to promote the intestinal development of juveniles has extremely important guiding significance for improving the success rate and quality of seedling cultivation. Summary of the Invention

[0004] The purpose of the present invention is to provide an additive for improving the survival, growth and intestinal development of juvenile marine fish. The main component includes melatonin, which can effectively promote the intestinal development of juvenile marine fish, improve the digestion and absorption ability, enhance the immunity, and is beneficial to the survival and growth of juvenile marine fish.

[0005] Specifically, an additive for improving the survival, growth and intestinal development of juvenile marine fish, the main component of which includes melatonin.

[0006] Preferably, the marine fish includes large yellow croaker.

[0007] In one embodiment, the additive comprises the following components in mass percentage:

[0008] Melatonin 0% - 2%, microcrystalline cellulose 0% - 2% and the balance α-starch, wherein melatonin is not zero.

[0009] Preferably, melatonin 1% - 2%, microcrystalline cellulose 0% - 1%.

[0010] Preferably, the analytical purity of the melatonin and microcrystalline cellulose is not less than 98% respectively.

[0011] Preferably, the particle size of the additive is 120 - 200 mesh.

[0012] The present invention also provides a method for preparing the above additive, which is obtained by mixing melatonin, microcrystalline cellulose and α - starch and pulverizing the resulting mixture.

[0013] The present invention also provides an aquatic feed for improving the survival, growth and intestinal development of juvenile marine fish.

[0014] Specifically, the aquatic feed for improving the survival, growth and intestinal development of juvenile marine fish comprises a basic feed and the above additive.

[0015] Preferably, the mass of the additive is 0.1% of the mass of the aquatic feed.

[0016] Beneficial effects:

[0017] The present invention provides the application of melatonin in products for improving the survival, growth and intestinal development of juvenile marine fish. Melatonin, a metabolite of tryptophan, has unique biological activity and can directly enter cells and be rapidly absorbed by juvenile marine fish. In vivo, melatonin exerts its effects through multiple mechanisms: it can not only promote the development of the juvenile fish intestine, but also significantly enhance the activity of digestive enzymes, thereby enhancing the digestion and absorption efficiency of nutrients. In addition, melatonin can also regulate the immune system of juvenile fish, enhance its immunity, and enable it to more effectively resist the invasion of pathogens. These effects synergistically improve the survival rate and growth performance of juvenile fish.

[0018] Based on the above advantages, the product provided by the present invention, in addition to containing melatonin, is also formulated with microcrystalline cellulose and α - starch. By reasonably proportioning melatonin with microcrystalline cellulose and α - starch or melatonin and α - starch, it effectively promotes the intestinal development of juvenile marine fish including juvenile large yellow croaker, improves the digestion and absorption ability, enhances the immunity, and is beneficial to the survival and growth of juvenile marine fish; meanwhile, the composition containing melatonin of the present invention has low raw material cost, high economy and strong operability. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments.

[0020] Figure 1 It is the detection result of the survival rate of juvenile large yellow croaker in each group in Application Example 1;

[0021] Figure 2 It is the detection result of the growth performance of juvenile large yellow croaker in each group in Application Example 1;

[0022] Figure 3 It is the detection result of the intestinal tissue morphology of juvenile large yellow croaker in each group in Application Example 1;

[0023] Figure 4 Results of detecting enzymes and genes related to intestinal development of juvenile large yellow croakers in each group in Application Example 1;

[0024] Figure 5 Results of detecting enzymes related to digestion and absorption in the intestines of juvenile large yellow croakers in each group in Application Example 1;

[0025] Figure 6 Results of detecting immune-related enzymes and nitric oxide content in the intestines of juvenile large yellow croakers in each group in Application Example 1. Detailed implementation manners

[0026] The present invention provides an application of melatonin in the preparation of a product for improving the survival, growth and intestinal development of juvenile marine fish.

[0027] In the present invention, the product preferably includes a feed additive and / or feed, and more preferably is a feed additive and feed. The melatonin in the present invention is a tryptophan metabolite with unique biological activity, which can directly enter cells and be rapidly absorbed by juvenile marine fish. In vivo, melatonin exerts its functions through multiple mechanisms: it can not only promote the development of the juvenile fish intestine, but also significantly enhance the activity of digestive enzymes, thereby enhancing the digestion and absorption efficiency of nutrients. In addition, melatonin can also regulate the immune system of juvenile fish, enhance its immunity, and enable it to more effectively resist the invasion of pathogens. These effects synergistically improve the survival rate and growth performance of juvenile fish.

[0028] Based on the above advantages, the present invention also provides an additive containing melatonin, and the additive includes components with the following mass percentages:

[0029] Melatonin 0% - 2%, microcrystalline cellulose 0% - 2% and the balance α-starch, wherein melatonin is not zero.

[0030] Calculated by mass percentage, the additive containing melatonin in the present invention includes 0% - 2% of melatonin, more preferably 1% - 2%. The analytical purity of the melatonin in the present invention is preferably not less than 98%. The melatonin in the present invention has the effect of improving the survival, growth and intestinal development of juvenile marine fish.

[0031] Based on the mass percentage of the melatonin, the additive in the present invention includes 0% - 2% of microcrystalline cellulose, more preferably 0 - 1%. The analytical purity of the microcrystalline cellulose in the present invention is preferably not less than 98%. The particle size of the microcrystalline cellulose in the present invention is preferably 120 - 200 mesh, more preferably 200 mesh.

[0032] Except for the melatonin and microcrystalline cellulose, the present invention makes up the remaining mass percentage with α-starch. In the present invention, the α-starch serves as a diluent.

[0033] The present invention effectively promotes the intestinal development of larvae of marine fish including larvae of large yellow croaker, improves the digestion and absorption ability, enhances the immunity, and is beneficial to the survival and growth of larvae of marine fish by reasonably proportioning melatonin with microcrystalline cellulose and α-starch or melatonin and α-starch.

[0034] The present invention also provides a preparation method of the above additive, including: mixing melatonin, microcrystalline cellulose and α-starch, and pulverizing the obtained mixture to obtain the additive.

[0035] The present invention mixes melatonin, microcrystalline cellulose and α-starch to form a uniform composite. Preferably, the present invention mixes melatonin and microcrystalline cellulose first and then mixes with α-starch. This mixing method adopts a step-by-step amplification mixing method, which can make each component mix more uniformly and helps to improve the uniformity of the mixture.

[0036] After obtaining the mixture, the present invention pulverizes the mixture to obtain the mixture containing melatonin. The pulverizing method of the present invention is preferably ultrafine pulverization. The particle size of the additive of the present invention is preferably 120-200 mesh, more preferably 200 mesh.

[0037] In the application of the additive of the present invention, the added mass is preferably 0.1% of the feed mass. The use method of the feed for improving the survival, growth and intestinal development of larvae of marine fish of the present invention is preferably feeding 7 times a day to satiation.

[0038] The present invention also provides a feed, including a basic feed and the above additive. In the feed of the present invention, the mass of the above additive is 0.1% of the feed mass. The present invention has no special limitation on the components and specific sources of the basic feed, and a basic feed with conventional components and commercially available sources in the art can be used. For example, the basic feed used in the examples of the present invention contains the following components in mass percentage: white fish meal 45.00%, krill meal 22.00%, yeast extract 3.50%, cuttlefish visceral powder 3.00%, high gluten flour 5.00%, α-starch 2.90%, sodium alginate 2.00%, compound vitamin 1.50%, compound mineral 1.00%, L-ascorbic acid-2-phosphate 0.20%, calcium dihydrogen phosphate 2.00%, mold inhibitor 0.05%, antioxidant 0.05%, choline chloride 0.20%, fish oil 6.50% and soy lecithin 5.00%.

[0039] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and examples, but they should not be construed as limiting the protection scope of the present invention.

[0040] Example 1

[0041] 1) Additive, consisting of the following components:

[0042] 0.5% melatonin, 1.5% microcrystalline cellulose and 98% α-starch, wherein the analytical purity of melatonin is ≥98%, and the analytical purity of microcrystalline cellulose is ≥98%;

[0043] Specifically, it is prepared according to 0.5 g of melatonin, 1.5 g of microcrystalline cellulose and 98 g of α-starch. The following examples or comparative examples are all prepared in this form and will not be repeated.

[0044] 2) Preparation method of the additive in 1) above, the steps are as follows:

[0045] Mix melatonin with microcrystalline cellulose, then mix with α-starch. After mixing evenly, it is ultramicro pulverized to 200 mesh to obtain the melatonin-containing composition.

[0046] Example 2

[0047] Differing from the example:

[0048] The additive consists of the following components: 1% melatonin, 1% microcrystalline cellulose and 98% high-gluten flour, wherein the analytical purity of melatonin is ≥98%, and the analytical purity of microcrystalline cellulose is ≥98%.

[0049] Example 3

[0050] Differing from the example:

[0051] The additive consists of the following components: 2% melatonin and 98% high-gluten flour, wherein the analytical purity of melatonin is ≥98%.

[0052] Comparative Example 1

[0053] 1) Additive, consisting of the following components:

[0054] 2% microcrystalline cellulose and 98% α-starch, wherein the analytical purity of microcrystalline cellulose is ≥98%;

[0055] 2) Preparation method of the additive in 1) above, the steps are as follows:

[0056] Mix microcrystalline cellulose with high-gluten flour, and after mixing evenly, it is ultramicro pulverized to 200 mesh to obtain.

[0057] Examples 4 to 7 and Comparative Example 2

[0058] Larval large yellow croaker feed, including a basic feed and an additive according to any one of Examples 1 to 3 and Comparative Example 1, wherein the feed of Example 4 contains the additive in Example 1 (denoted as Feed 2), the feed of Example 5 contains the additive in Example 2 (denoted as Feed 3), the feed of Example 6 contains the additive in Example 3 (denoted as Feed 4), and the feed of Comparative Example 2 contains the additive in Comparative Example 1 (denoted as Feed 1). The specific feed formula is shown in Table 1. The additive in any one of Examples 4 to 7 and Comparative Example 2 accounts for 0.1% of the total mass of the feed.

[0059] Table 1 Formulas of feeds in Examples 4 to 7 and Comparative Example 2

[0060]

[0061] 1 Compound vitamins (mg / kg): retinol acetate, 32; α-tocopherol, 240; menadione, 10; vitamin B1, 25; pyridoxine hydrochloride, 20; vitamin B12, 10; riboflavin, 45; pantothenic acid, 60; vitamin D3, 5; folic acid, 20; nicotinic acid, 200; biotin, 60; inositol, 800; microcrystalline cellulose, 13473.

[0062] 2 Compound minerals: MgSO4·7H2O, 1200; FeSO4·H2O, 80; ZnSO4·H2O, 50; CuSO4·5H2O, 10; MnSO4·H2O, 45; CoCl2·6H2O, 50; Na2SeO3, 20; H2CaIO4, 60; zeolite powder, 13485.

[0063] Application Example 1

[0064] Application of the larval large yellow croaker feeds in Examples 4 to 7 and Comparative Example 2 in the breeding of larval large yellow croaker is as follows:

[0065] For the experimental larval large yellow croaker, 36,000 larval large yellow croaker with an initial body weight of 4.99 ± 0.48 mg were selected and randomly divided into 4 groups, with 3 replicates in each group and 3000 fish in each replicate. A 30-day feeding growth experiment was carried out. The breeding water temperature was 23 - 25 °C, the pH was 7.8 - 8.2, and the salinity was 22 - 24‰. The feeds of Examples 4 to 7 and Comparative Example 2 were used for feeding, and they were fed 7 times a day, all to satiation. The feeding times were 05:30, 08:30, 11:30, 14:30, 17:30, 20:30, and 23:30.

[0066] After 30 days of breeding, the intestines and intestinal segments of the larval large yellow croaker in each group were collected and the following detections were carried out, specifically:

[0067] When performing histological analysis of the intestine, tissue samples are first subjected to a series of delicate pretreatment steps, including fixation, dehydration, clearing, infiltration with paraffin wax, and embedding. Subsequently, the samples are sectioned into thin slices, and the operations of picking up the sections and baking them are completed in sequence. Next, the sections are stained with the classic hematoxylin-eosin (H&E). After staining, the sections are sealed with neutral resin to ensure the stability and integrity of the samples. When all the above steps are completed, the samples are placed under a microscope for observation. Initially, each tissue section is initially examined at a low magnification to obtain a macroscopic overview of the tissue structure and accurately locate the key areas that require further detailed observation. Subsequently, ImageJ software is used to process all the images to evaluate the fold height, intestinal epithelial cell height, and muscle thickness.

[0068] The determination of the expression levels of genes related to intestinal development was carried out by quantitative q-PCR analysis. ChamQ Universal SYBR qPCR Master Mix (Novoprotein Scientific Inc.) was used to perform q-PCR detection on the following genes respectively to obtain the expression levels of mRNA, specifically: Proliferating cell nuclear antigen (pcna) and Ornithine decarboxylase (odc), with beta-actin (β-actin) as the internal reference gene. q-PCR primers were designed according to the nucleotide sequences of large yellow croaker (Table 2).

[0069] Table 2 q-PCR primer sequences

[0070]

[0071]

[0072] For the extraction of the intestinal brush border membrane, first, the intestinal segments are homogenized in a 2 mL mixed solution containing 50 mmol / L mannitol and 2 mmol / L Tris (volume ratio 1:1). Subsequently, 1 mL of 0.1 mol / L CaCl2 solution is added to the homogenate. Then, the mixture is centrifuged at 9000×g for 10 minutes, and the supernatant is collected. After that, the supernatant is centrifuged at 34000×g for 20 minutes, and the supernatant is discarded. Finally, the obtained precipitate rich in intestinal brush border membrane is resuspended in 1 mL of a mixed solution composed of 0.1 mol / L KCl, 5 mmol / L Tris-Hepes (pH 7.5), and 1 mmol / L DTT (volume ratio 1:1:1). This method can effectively isolate the intestinal brush border membrane.

[0073] The activity of leucine aminopeptidase (LAP) was determined using leucine-p-nitroaniline as the substrate. The specific procedure was as follows: In a centrifuge tube, first add 1.5 mL of phosphate buffer (20 mmol / L, pH 7.2), then add 100 μL of leucine-p-nitroaniline substrate solution, and finally add 100 μL of the sample preheated to 30 °C. After starting the reaction, use a spectrophotometer to measure the absorbance of the released p-nitroaniline at 405 nm. According to the molar absorption coefficient of p-nitroaniline (8800 L·mol-1·cm-1), calculate the hydrolysis rate of the substrate to reflect the enzyme activity.

[0074] Alkaline phosphatase, lipase, α-amylase, trypsin, Na + K + –ATPase, creatine kinase, acid phosphatase, nitric oxide content, nitric oxide synthase, and lysozyme were all detected using commercial kits, and the specific purchase source was Nanjing Jiancheng Bioengineering Institute; the names of the kits were as follows:

[0075] Alkaline phosphatase (ALP / AKP) assay kit (microplate method) (A059-2-2), lipase assay kit (microplate method) (A054-2-1), α-amylase (AMS) test kit (starch-iodine colorimetric method) (C016-1-1), trypsin assay kit (ultraviolet colorimetric method) (A080-2-2), ultra-trace Na + K + –ATPase (for tissues and normal cells) kit (A070-2-2), creatine kinase (CK) assay kit (colorimetric method), acid phosphatase (ACP) assay kit (microplate method) (A060-2-2), nitric oxide (NO) assay kit (enzymatic method) colorimetric method (A012-1-2), nitric oxide synthase (NOS) typing test kit (colorimetric method) (A014-1-2), and lysozyme (LZM) test kit (A050-1-1).

[0076] The experimental data were statistically analyzed using one-way analysis of variance in SPSS 17.0. After significant differences were found, Tukey's multiple comparisons were performed, and the significance level was P < 0.05. The obtained experimental data were expressed as means ± standard error (means ± S.E.M., n = 3).

[0077] The results are as follows:

[0078] 1) In terms of survival rate and growth performance, compared with Feed 1, Feed 3 and Feed 4 significantly increased the survival rate of juvenile large yellow croaker (P < 0.05) ( Figure 1 ). At the same time, compared with Feed 1, Feed 3 and Feed 4 significantly increased the final body weight and specific growth rate of juvenile large yellow croaker (P < 0.05) (Figure 2 A and 2B). This indicates that the use of melatonin-containing additives can improve the survival and growth of juvenile Larimichthys crocea.

[0079] 2) In terms of intestinal development, compared with Feed 1 and Feed 2, Feed 3 and Feed 4 significantly increased the intestinal fold height of juvenile Larimichthys crocea (P < 0.05) ( Figure 3 A and 3B). At the same time, compared with Feed 1, Feed 3 and Feed 4 significantly increased the activities of alkaline phosphatase and leucine aminopeptidase in the intestinal brush border of juvenile Larimichthys crocea (P < 0.05) ( Figure 4 A and 4B). In addition, compared with Feed 1, Feed 2, Feed 3 and Feed 4 significantly increased the expression level of the gene (pcna) related to intestinal development in juvenile Larimichthys crocea (P < 0.05) ( Figure 4 C). At the same time, compared with Feed 1, Feed 2 and Feed 3 significantly increased the expression level of the gene (odc) related to intestinal development in juvenile Larimichthys crocea (P < 0.05) ( Figure 4 D). This indicates that the use of melatonin-containing additives can promote the intestinal development of juvenile Larimichthys crocea.

[0080] 3) In terms of digestion and absorption, compared with Feed 1, Feed 2 and Feed 3 significantly increased the lipase activity in the intestine of juvenile Larimichthys crocea (P < 0.05) ( Figure 5 A). At the same time, compared with Feed 1 and Feed 2, Feed 3 and Feed 4 significantly increased the trypsin activity in the intestine of juvenile Larimichthys crocea (P < 0.05) ( Figure 5 C). In addition, compared with Feed 1, Feed 3 and Feed 4 significantly increased the creatine kinase activity in the intestinal brush border of juvenile Larimichthys crocea (P < 0.05) ( Figure 5 D). At the same time, compared with Feed 1, Feed 4 significantly increased the Na + K + –ATPase activity in the intestine of juvenile Larimichthys crocea (P < 0.05) ( Figure 5 E). This indicates that the use of melatonin-containing additives can improve the digestion and absorption ability of juvenile Larimichthys crocea.

[0081] 4) In terms of immunity, compared with Feed 1, Feed 3 significantly increased the activities of lysozyme and acid phosphatase in the intestine of juvenile Larimichthys crocea (P < 0.05) ( Figure 6 A and 6B). At the same time, compared with Feed 1, Feed 3 and Feed 4 significantly increased the content of nitric oxide in the intestine of juvenile Larimichthys crocea (P < 0.05) ( Figure 6 C). In addition, compared with Feed 1, Feed 3 and Feed 4 significantly increased the total nitric oxide synthase activity in the intestine of juvenile Larimichthys crocea (P < 0.05) ( Figure 5 D). At the same time, compared with Feed 1, Feed 4 significantly increased the inducible nitric oxide synthase activity in the intestine of juvenile Larimichthys crocea (P < 0.05) ( Figure 5E). This indicates that the use of the melatonin-containing additive can enhance the immunity of juvenile Larimichthys crocea.

[0082] From the above results, it can be concluded that the additive and feed containing melatonin according to the present invention can effectively promote the intestinal development of juvenile marine fish including juvenile Larimichthys crocea, improve the digestion and absorption ability, enhance the immunity, and improve the survival and growth of juvenile marine fish.

[0083] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, rather than all embodiments. People can also obtain other embodiments based on these embodiments without creative efforts, and these embodiments all fall within the protection scope of the present invention.

Claims

1. An additive for improving the survival, growth and intestinal development of marine fish larvae, characterized in that, The main components include melatonin.

2. The additive according to claim 1, characterized in that, The seawater fish includes large yellow croaker.

3. The additive according to claim 1, characterized in that, It includes components with the following mass percentages: Melatonin 0% - 2%, microcrystalline cellulose 0% - 2% and the balance α-starch, wherein melatonin is not zero.

4. The additive according to claim 3, characterized in that, The mass percentage of the melatonin is 0.5% - 2%.

5. The additive according to claim 3 or 4, characterized in that, The analytical purities of the melatonin and microcrystalline cellulose are not less than 98% respectively.

6. The additive according to any one of claims 1-5, characterized in that, The particle size of the additive is 120 - 200 mesh.

7. The preparation method of the additive according to any one of claims 1-6, characterized in that, Mix melatonin, microcrystalline cellulose and α-starch, and pulverize the obtained mixture to obtain the product.

8. Feed for improving the survival, growth and intestinal development of marine fish larvae, characterized in that, The components include a basic feed and the additive according to any one of claims 1 - 6.

9. The feed according to claim 8, characterized in that, The mass of the additive is 0.1% of the mass of the aquatic feed.

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