Vibrio-preventing and growth-promoting feed for prawns

Abstract

The invention relates to the technical field of feeds, in particular to a vibrio-preventing and growth-promoting feed for prawns, which comprises the following components: a basal feed and scutellaria baicalensis flavone, and 100-400mg of scutellaria baicalensis flavone is mixed with the basal feed per 1kg of the basal feed. In the feed, the addition of the scutellaria baicalensis flavone can improve the vibrio infection resistance of the prawns, promote the growth of the prawns, maintain the stability of intestinal flora, and greatly improve the immunity of the prawns. And the dosage proportion of each component in the basal feed is reasonable, so that nutritional ingredients required by prawn production are met.

Description

Technical Field

[0001] The present invention relates to the technical field of feeds, and in particular to an anti-vibrio and growth-promoting feed for penaeid shrimps. Background Art

[0002] Vibriosis is one of the most common and harmful diseases affecting the penaeid shrimp aquaculture industry. Its symptoms generally include red legs, yellow gills, rotten tails, rotten eyes, shell ulcers, muscle turbidity, etc., often accompanied by septicemia. Vibrio parahaemolyticus is the earliest discovered pathogenic vibrio of penaeid shrimps. For many years, antibiotics have been mainly used to treat vibriosis, but antibiotic treatment has brought a series of serious consequences, mainly the generation of drug-resistant strains, water pollution, and damage to the immunity of shrimps. Therefore, it is imperative to explore new treatment methods.

[0003] Scutellaria baicalensis contains various flavonoid compounds such as baicalin, baicalein, wogonoside, and wogonin. Baicalin flavonoids have antibacterial, anti-inflammatory, antiviral, antioxidant, antitumor, and immune-enhancing effects on the body. At present, baicalin flavonoids are mainly used to improve the production performance, antioxidant effect, and inflammation treatment of livestock and poultry, and there is no report on its research in aquatic animals. In view of the excellent biological activity of baicalin flavonoids, in order to improve the immune prevention of the self-immunity of shrimps, an anti-vibrio and growth-promoting feed for penaeid shrimps has been developed. Summary of the Invention

[0004] The technical problem to be solved by the present invention is: aiming at the deficiencies of the prior art, to provide an anti-vibrio and growth-promoting feed for penaeid shrimps, which can improve the ability of penaeid shrimps to resist vibrio infection, promote the growth of penaeid shrimps, maintain the stability of intestinal flora, and greatly improve the immunity of penaeid shrimps.

[0005] To solve the above technical problem, the technical solution of the present invention is:

[0006] An anti-vibrio and growth-promoting feed for penaeid shrimps, the feed comprising the following components: a basic feed and baicalin flavonoids, wherein the baicalin flavonoids are mixed with the basic feed according to an addition amount of 100 - 400 mg per 1 kg of the basic feed.

[0007] As a preferred technical solution, the baicalin flavonoids are mixed with the basic feed according to an addition amount of 400 mg per 1 kg of the basic feed.

[0008] As an improved technical solution, the basic feed comprises the following components by weight: 200 g of fish meal, 300 g of wheat gluten, 200 g of wheat flour, 180 g of cellulose, 25 g of fish oil, 25 g of soybean oil, 20 g of phospholipids, 20 g of binder, 10 g of choline chloride, 10 g of compound vitamins, and 10 g of compound minerals.

[0009] As an improved technical solution, per 1 kg of the basal feed, the compound vitamins include the following components by weight: riboflavin 45 mg, thiamine 25 mg, vitamin K3 10 mg, inositol 800 mg, pyridoxine hydrochloride 20 mg, vitamin B12 0.1 mg, calcium pantothenate 60 mg, biotin 1.2 mg, vitamin A 32 mg, vitamin D 5 mg, niacin 200 mg, folic acid 20 mg, and vitamin E 120 mg.

[0010] As an improved technical solution, per 1 kg of the basal feed, the compound minerals include the following components by weight: KI 0.8 mg, NaF 2 mg, Fe 2 (SO 4 ) 3 80 mg, ZnSO 4 50 mg, CoCl 2 ·6H 2 O 50 mg, CuSO 4 ·5H 2 O 10 mg, MgSO 4 200 mg, Nacl 100 mg, Ca(H 2 PO 4 ) 2 3000 mg.

[0011] As an improved technical solution, in per 1 kg of the basal feed, the crude protein content in the fish meal is 689.9 g / kg dry matter, and the crude fat content is 78.1 g / kg dry matter; the crude protein content in the wheat gluten is 790.5 g / kg dry matter, and the crude fat content is 1.8 g / kg dry matter; the crude protein content in the wheat flour is 165.0 g / kg dry matter, and the crude fat content is 15.8 g / kg dry matter.

[0012] After adopting the above technical solution, the beneficial effects of the present invention are as follows:

[0013] The present invention uses the basal feed and baicalein flavonoids as the components of the shrimp feed, and the baicalein flavonoids are mixed with the basal feed at a dosage of 100 - 400 mg per 1 kg of the basal feed. The addition of baicalein flavonoids can improve the ability of shrimp to resist Vibrio infection, promote the growth of shrimp, maintain the stability of the intestinal flora, and greatly improve the immunity of shrimp. Moreover, the dosage ratio of each component in the basal feed is reasonable, meeting the nutritional components required for shrimp production. Description of the Drawings

[0014] Figure 1 It is a picture of the external appearance of shrimp after being infected with Vibrio;

[0015] Figure 2Graph for investigating the cumulative mortality rate of penaeid shrimp and the protection rate of additives;

[0016] Figure 3 Schematic diagram of the pancreatic tissue structure of penaeid shrimp;

[0017] Figure 4 Schematic diagram of the intestinal tissue structure of penaeid shrimp;

[0018] Figure 5 Graph for investigating the clearance of Vibrio in the hepatopancreas of penaeid shrimp;

[0019] Figure 6 Graph for investigating the immune enzyme activity of penaeid shrimp;

[0020] Figure 7 Graph for investigating the blood biochemical indexes of penaeid shrimp;

[0021] Figure 8 Graph for investigating Proteobacteria, Bacteroidetes, and Firmicutes in the shrimp intestine. Detailed implementation manners

[0022] In order to make the objectives, technical solutions, and advantages of the present invention clearer and more understandable, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0023] Example 1

[0024] An anti-Vibrio and growth-promoting feed for penaeid shrimp, comprising the following components: a basic feed and baicalein flavone, wherein the baicalein flavone is mixed with the basic feed at an addition amount of 100 mg per 1 kg of the basic feed.

[0025] The basic feed comprises the following components by weight: 200 g of fish meal, 300 g of wheat gluten, 200 g of wheat flour, 180 g of cellulose, 25 g of fish oil, 25 g of soybean oil, 20 g of phospholipid, 20 g of binder, 10 g of choline chloride, 10 g of compound vitamin, and 10 g of compound mineral.

[0026] Among them, the compound vitamin in every 1 kg of the basic feed comprises the following components by weight: 45 mg of riboflavin, 25 mg of thiamine, 10 mg of vitamin K3, 800 mg of inositol, 20 mg of pyridoxine hydrochloride, 0.1 mg of vitamin B12, 60 mg of calcium pantothenate, 1.2 mg of biotin, 32 mg of vitamin A, 5 mg of vitamin D, 200 mg of niacin, 20 mg of folic acid, and 120 mg of vitamin E.

[0027] Among them, the compound mineral in every 1 kg of the basic feed comprises the following components by weight: 0.8 mg of KI, 2 mg of NaF, Fe 2 (SO4 ) 3 80 mg, ZnSO 4 50 mg, CoCl 2 ·6H 2 O 50 mg, CuSO 4 ·5H 2 O 10 mg, MgSO 4 200 mg, Nacl 100 mg, Ca(H 2 PO 4 ) 2 3000 mg.

[0028] Among them, the content of crude protein in fish meal per 1 kg of basal feed is 689.9 g / kg dry matter, and the content of crude fat is 78.1 g / kg dry matter; the content of crude protein in wheat gluten is 790.5 g / kg dry matter, and the content of crude fat is 1.8 g / kg dry matter; the content of crude protein in wheat flour is 165.0 g / kg dry matter, and the content of crude fat is 15.8 g / kg dry matter.

[0029] Example 2

[0030] Different from Example 1, for the vibrio - preventing and growth - promoting feed for prawns, baicalein is mixed with the basal feed at an addition amount of 200 mg per 1 kg of basal feed.

[0031] Example 3

[0032] Different from Example 1, for the vibrio - preventing and growth - promoting feed for prawns, baicalein is mixed with the basal feed at an addition amount of 400 mg per 1 kg of basal feed.

[0033] In order to better prove that the feed of the present invention can improve the ability of prawns to resist vibrio infection, promote the growth of prawns, maintain the stability of intestinal flora, and greatly improve the immunity of prawns, the following test examples are given.

[0034] Test Example 1 Investigation of the aquaculture water quality of Litopenaeus vannamei

[0035] A 30 - day aquaculture experiment was carried out on Litopenaeus vannamei using the feed in Example 3. After detection, it was found that all the conventional water quality parameters of the aquaculture water body of Litopenaeus vannamei were maintained within the normal range, with the temperature being 26.38 - 28.68 °C, the salinity being 19.44‰ - 20.80‰, the pH being 7.81 - 8.22, and the DO being 2.2 - 7.3. There were no significant differences in parameters such as temperature, salinity, pH, and DO among the test groups. The above results indicate that this feed has no obvious impact on the aquaculture water quality of Litopenaeus vannamei.

[0036] Test Example 2 Investigation of the effects on the growth, survival, and appearance of Litopenaeus vannamei

[0037] (1) Investigation on the growth of penaeid shrimp

[0038] Select 360 healthy Litopenaeus vannamei with an initial weight of 0.57 ± 0.06 g per shrimp, and then randomly divide them into 4 groups. Those without adding the feed of the present invention are used as the control group, those adding the feed of Example 1 are used as Experimental Group 1, those adding the feed of Example 2 are used as Experimental Group 2, and those adding the feed of Example 3 are used as Experimental Group 3, and then carry out 30 days of cultivation. The specific test results are shown in Table 1.

[0039] Table 1

[0040]

[0041] It can be found from the data in Table 1 that the growth-related indexes such as the body weight gain, relative weight gain rate, specific growth rate and feed coefficient of the penaeid shrimp in Experimental Groups 1 - 3 increase significantly, and are significantly better than those of the control group. In addition, it can also be found that the growth-related indexes of the penaeid shrimp in Experimental Group 3 are significantly better than those in Experimental Group 1 and Experimental Group 2.

[0042] (2) Investigation on the appearance and survival of penaeid shrimp

[0043] Randomly divide 450 healthy Litopenaeus vannamei with an initial weight of 0.57 ± 0.06 g into 5 groups. Among them, the healthy group without Vibrio infection and not fed with the feed of the present invention is used as the blank control group, the group infected with Vibrio and not fed with the feed of the present invention is used as the control group, and the groups infected with Vibrio and fed with the feeds of Example 1, Example 2 and Example 3 are respectively corresponding to Experimental Group 1, Experimental Group 2 and Experimental Group 3, and carry out 10 days of feeding. During the test period, observe the appearance signs, swimming conditions, feeding conditions and hepatopancreas appearance of the penaeid shrimp every morning when feeding the feed. Measure the body weight of the penaeid shrimp before the start (0 d) and at the end (30 d) of the cultivation test, record the daily feed amount fed to the penaeid shrimp, and calculate the specific growth rate (SGR), relative weight gain rate (WG) and feed coefficient (FCR) of the penaeid shrimp.

[0044] SGR (% / d) = 100×[(lnWt – lnW0)] / t

[0045] WG (%) = [(Wt – W0) / W0]×100%;

[0046] FCR (%) = FI / W

[0047] In the formula: Wt is the body weight of the shrimp at the end of the test (g), W0 is the body weight of the shrimp at the beginning of the test (g); t is the test time (d); FI is the dry matter of the feed ingested during the test period (g); W is the weight increase of the penaeid shrimp during the cultivation period (g).

[0048] After counting the number of dead shrimp in each group within ten days after Vibrio infection (with no obvious response when touched), calculate the cumulative mortality rate of shrimp in each test group and the protection rate (RPS) of the additive.

[0049] Cumulative mortality rate (%) = Cumulative number of dead shrimp / Initial number of shrimp × 100%

[0050] RPS = (1 - Cumulative mortality rate of each group / Cumulative mortality rate of the infected group only) × 100%

[0051] During the infection test stage, on the 1st day after infection (P1d), except for the blank control group, the shrimp in Control Group 1, Test Group 1, and Test Group 2 showed infection symptoms. The color of the hepatopancreas became lighter and paler, the boundary clarity decreased, and severe empty intestine and empty stomach phenomena occurred. However, the above symptoms in Test Group 3 were significantly milder than those in other groups. On the 10th day after infection (P10d), the hepatopancreas of the blank control group had clear boundaries, the intestine was full, and the body color was translucent. Only the control group had a pale body color, empty intestine and empty stomach, and the hepatopancreas was atrophied. The boundaries of the hepatopancreas of the shrimp in Test Group 1, Test Group 2, and Test Group 3 were significantly clearer than those in the infected group only, the body color was restored, and the empty intestine and empty stomach phenomena were improved, and the degree of improvement was positively correlated with the drug dosage (for details, see Figure 1 ).

[0052] Almost no shrimp died in the blank control group. Compared with the control group, the cumulative mortality rate of shrimp in Test Group 1 to Test Group 3 after Vibrio infection was significantly reduced (P < 0.05). Among Test Group 1, Test Group 2, and Test Group 3, there were significant differences in the cumulative mortality rate (P < 0.05). As the dose of baicalein flavonoids in the feed increased, the cumulative mortality rate gradually decreased. There was no significant difference in the cumulative mortality rate among groups with the same dose of baicalein flavonoids (P > 0.05) (for details, see Figure 2 ). In summary, the shrimp in the high-dose baicalein flavonoids group had the mildest symptoms and the lowest cumulative mortality rate after Vibrio infection.

[0053] Investigation on the effects of Test Example 3 on the hepatopancreas and intestine of Litopenaeus vannamei

[0054] 450 healthy Litopenaeus vannamei with an initial weight of 0.57 ± 0.06 g were randomly divided into 5 groups. Among them, the healthy group that was not infected with Vibrio and not fed with the feed of the present invention was used as the blank control group, the group that was infected with Vibrio and not fed with the feed of the present invention was used as the control group, and the groups that were infected with Vibrio and fed with the feeds of Example 1, Example 2, and Example 3 were respectively corresponding to Test Group 1, Test Group 2, and Test Group 3. Among them, the hepatopancreas of the shrimp was investigated for 30 days of feeding, and the intestine of the shrimp was investigated for 10 days of feeding. At 0d, 30d, P1d, and P10d, the hepatopancreas and intestinal tissues of 2 shrimp were randomly taken from each test group, fixed with 4% paraformaldehyde solution, made into sections, and observed under a microscope after HE staining.

[0055] It can be found through experiments that on the 0th day of cultivation (corresponding to 3-0 in the attached figure), and on the 30th day (corresponding to 3-1 in the attached figure), the hepatopancreas tissue of each group of shrimps was in good condition, the hepatopancreas tubules were arranged neatly, the lumen was clear, the structure was complete, the hepatopancreas epithelial cells were complete and full; on the 1st day after infection (corresponding to 3-2 in the attached figure), only in the control group, the hepatopancreas tubules were arranged loosely and the structure was damaged, the epithelial cells of the hepatopancreas tubules ruptured, and the nuclear mass overflowed; the hepatopancreas tubules of the remaining experimental groups 1, 2, and 3 all showed the same tissue damage, and the degree of damage decreased with the increase of the drug dosage. Therefore, it can be concluded that when the feed of the present invention is added to cultivate shrimps, the recovery of the hepatopancreas tissue is better than that of the infected group only, the arrangement and structural integrity of the hepatopancreatic tubules are gradually improved, the lumen becomes gradually clear and obvious, the number of epithelial cells of the hepatopancreatic tubules increases, and the structural integrity is enhanced. And with the increase of the dosage of baicalein flavonoids in the feed, the improvement of the hepatopancreas tissue gradually increases (for details, see Figure 3 In the figure, A: control group, B: infected group only, G: baicalein flavonoids 100mg / kg, H: baicalein flavonoids 200mg / kg, I: baicalein flavonoids 400mg / kg. a is the lumen of the hepatopancreatic tubule, b is the epithelial cell of the hepatopancreatic tubule). Figure 3 It can be found through experiments that on the 0th day of cultivation (corresponding to 4-0 in the attached figure), and on the 30th day (corresponding to 4-1 in the attached figure), the intestinal tissue morphology of each group of shrimps was normal, the intestinal epithelial cells were tightly connected, and the microvilli were arranged neatly. On the 1st day after infection (corresponding to 4-2 in the attached figure), only in the control group, the intestinal epithelial cells exfoliated and the microvilli disappeared; the intestinal tissues of experimental groups 1, 2, and 3 all showed varying degrees of epithelial cell exfoliation, microvilli exfoliation and shortening, and the degree of damage decreased with the increase of the drug dosage. Therefore, it can be concluded that when the feed of the present invention is added to cultivate shrimps, the recovery of the intestinal tissue is better than that of the control group, the number of epithelial cells in the intestinal tissue increases, the length and number of microvilli also increase, the neatness of the microvilli arrangement is gradually improved, and the structural integrity is enhanced. And with the increase of the dosage of the additive, the improvement of the intestinal tissue gradually increases (for details, see Figure 3 , it should be noted that: Figure 3 In the figure, A: control group, B: infected group only, G: baicalein flavonoids 100mg / kg, H: baicalein flavonoids 200mg / kg, I: baicalein flavonoids 400mg / kg. a is the intestinal epithelial microvilli).

[0056] It can be found through experiments that on the 0th day of cultivation (corresponding to 4-0 in the attached figure), and on the 30th day (corresponding to 4-1 in the attached figure), the intestinal tissue morphology of each group of shrimps was normal, the intestinal epithelial cells were tightly connected, and the microvilli were arranged neatly. On the 1st day after infection (corresponding to 4-2 in the attached figure), only in the control group, the intestinal epithelial cells exfoliated and the microvilli disappeared; the intestinal tissues of experimental groups 1, 2, and 3 all showed varying degrees of epithelial cell exfoliation, microvilli exfoliation and shortening, and the degree of damage decreased with the increase of the drug dosage. Therefore, it can be concluded that when the feed of the present invention is added to cultivate shrimps, the recovery of the intestinal tissue is better than that of the control group, the number of epithelial cells in the intestinal tissue increases, the length and number of microvilli also increase, the neatness of the microvilli arrangement is gradually improved, and the structural integrity is enhanced. And with the increase of the dosage of the additive, the improvement of the intestinal tissue gradually increases (for details, see Figure 4 In the figure), and on the 30th day (corresponding to 4-1 in the attached figure), the intestinal tissue morphology of each group of shrimps was normal, the intestinal epithelial cells were tightly connected, and the microvilli were arranged neatly. On the 1st day after infection (corresponding to 4-2 in the attached figure), only in the control group, the intestinal epithelial cells exfoliated and the microvilli disappeared; the intestinal tissues of experimental groups 1, 2, and 3 all showed varying degrees of epithelial cell exfoliation, microvilli exfoliation and shortening, and the degree of damage decreased with the increase of the drug dosage. Therefore, it can be concluded that when the feed of the present invention is added to cultivate shrimps, the recovery of the intestinal tissue is better than that of the control group, the number of epithelial cells in the intestinal tissue increases, the length and number of microvilli also increase, the neatness of the microvilli arrangement is gradually improved, and the structural integrity is enhanced. And with the increase of the dosage of the additive, the improvement of the intestinal tissue gradually increases (for details, see Figure 4 In the figure), and on the 30th day (corresponding to 4-1 in the attached figure), the intestinal tissue morphology of each group of shrimps was normal, the intestinal epithelial cells were tightly connected, and the microvilli were arranged neatly. On the 1st day after infection (corresponding to 4-2 in the attached figure), only in the control group, the intestinal epithelial cells exfoliated and the microvilli disappeared; the intestinal tissues of experimental groups 1, 2, and 3 all showed varying degrees of epithelial cell exfoliation, microvilli exfoliation and shortening, and the degree of damage decreased with the increase of the drug dosage. Therefore, it can be concluded that when the feed of the present invention is added to cultivate shrimps, the recovery of the intestinal tissue is better than that of the control group, the number of epithelial cells in the intestinal tissue increases, the length and number of microvilli also increase, the neatness of the microvilli arrangement is gradually improved, and the structural integrity is enhanced. And with the increase of the dosage of the additive, the improvement of the intestinal tissue gradually increases (for details, see Figure 4 In the figure), only in the control group, the intestinal epithelial cells exfoliated and the microvilli disappeared; the intestinal tissues of experimental groups 1, 2, and 3 all showed varying degrees of epithelial cell exfoliation, microvilli exfoliation and shortening, and the degree of damage decreased with the increase of the drug dosage. Therefore, it can be concluded that when the feed of the present invention is added to cultivate shrimps, the recovery of the intestinal tissue is better than that of the control group, the number of epithelial cells in the intestinal tissue increases, the length and number of microvilli also increase, the neatness of the microvilli arrangement is gradually improved, and the structural integrity is enhanced. And with the increase of the dosage of the additive, the improvement of the intestinal tissue gradually increases (for details, see Figure 4 , it should be noted that A: control group, B: infected group only, G: baicalein flavonoids 100mg / kg, H: baicalein flavonoids 200mg / kg, I: baicalein flavonoids 400mg / kg. a is the intestinal epithelial microvilli).

[0057] In summary, when used as a feed additive, Scutellaria flavonoids have a certain growth-promoting effect on Litopenaeus vannamei and can significantly improve the ability of the shrimp to resist Vibrio infection. By comparing the experimental effects of different doses of Scutellaria flavonoids, the recommended dose of Scutellaria flavonoids is suggested to be 400 mg / kg.

[0058] Experimental Example 4 Investigation of the clearance rate of Vibrio in Litopenaeus vannamei

[0059] 450 healthy Litopenaeus vannamei with an initial weight of 0.57 ± 0.06 g were randomly divided into 5 groups. Among them, the healthy group that was not infected with Vibrio and not fed with the feed of the present invention was used as the blank control group, the group infected with Vibrio and not fed with the feed of the present invention was used as the control group, and the groups infected with Vibrio and fed with the feeds of Example 1, Example 2, and Example 3 were respectively corresponding to Experimental Group 1, Experimental Group 2, and Experimental Group 3, and they were fed for 10 days. Randomly take 2 shrimps from each experimental group at 0 d, 30 d, P1 d, and P10 d, collect the hepatopancreas and grind it into a homogenate by adding sterile PBS solution. Take 100 μL of the mixed solution and spread it on a TCBS agar plate, and incubate it at 28 °C for 24 hours for Vibrio colony counting.

[0060] During the Vibrio infection process in this experiment, the infected shrimps showed stronger antibacterial activity than the shrimps in the control group. In addition, compared with the control group, the remaining Vibrio in the hepatopancreas of the shrimps infected with Vibrio in Experimental Group 1, Experimental Group 2, and Experimental Group 3 was significantly reduced. In Experimental Groups 1 - 3, with different doses of Scutellaria flavonoids, there were significant differences in the clearance rate of Vibrio in the hepatopancreas (P < 0.05). As the dose of Scutellaria flavonoids increased, the concentration of Vibrio in the hepatopancreas gradually decreased (for details, see Figure 5 , where it should be noted that: a in the attached figure represents the blank control group, b represents the group only infected, c represents the dosage of 100 mg / kg of Scutellaria flavonoids corresponding to 1 day of infection, d represents the dosage of 200 mg / kg of Scutellaria flavonoids corresponding to 1 day of infection, e represents the dosage of 400 mg / kg of Scutellaria flavonoids corresponding to 1 day of infection; in accordance with the above order, they were infected for 10 days in turn and compared with the attached figure).

[0061] Experimental Example 5 Investigation of the effect on the immune enzyme activity of Litopenaeus vannamei

[0062] 450 healthy Litopenaeus vannamei with an initial weight of 0.57 ± 0.06 g were randomly divided into 5 groups. Among them, the healthy group without Vibrio infection and not fed with the feed of the present invention was used as the blank control group, the group infected with Vibrio and not fed with the feed of the present invention was used as the control group, and the groups infected with Vibrio and fed with the feeds of Example 1, Example 2 and Example 3 were respectively used as Experimental Group 1, Experimental Group 2 and Experimental Group 3, and fed for 10 days. At 0 d, 30 d, P1 d, and P10 d, the hepatopancreas of 2 shrimps was randomly taken from each experimental group, added with physiological saline and ground into a homogenate, and the supernatant was taken after centrifugation. Kits from Nanjing Jiancheng Bioengineering Institute were used to measure the activities of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), acid phosphatase (ACP), alkaline phosphatase (AKP), and lysozyme (LZM). The activity of phenol oxidase (PO) was measured by spectrophotometry using L-3,4-dihydroxyphenylalanine (L-DOPA) as the substrate.

[0063] It can be found through the experiment that the activities of ACP, AKP, LZM, and SOD showed a trend of first increasing and then decreasing, reaching the peak on the 1st day after infection (P1 d), the activity of GSH-Px showed a continuously increasing trend, reaching the peak on the 10th day after infection (P10 d), and the activity of PO showed a trend of first decreasing and then increasing, reaching the lowest value on the 1st day after infection (P1 d). At each sampling time point, compared with the control group, the activities of ACP, AKP, GSH-Px, LZM, PO, and SOD in Experimental Groups 1-3 increased to varying degrees, and the increase level was positively correlated with the dose (see details in Figure 6 , where it should be noted that Figure 6-1 In Figure 6-6, a represents the blank control group, b represents the group with a baicalein flavone dosage of 100 mg / kg, c represents the group with a baicalein flavone dosage of 200 mg / kg, d represents the group with a baicalein flavone dosage of 400 mg / kg, and e represents the group only infected with Vibrio).

[0064] Effect of Test Example 6 on the blood biochemical indexes of Litopenaeus vannamei

[0065] 450 healthy Litopenaeus vannamei with an initial weight of 0.57 ± 0.06 g were randomly divided into 5 groups. Among them, the healthy group without Vibrio infection and not fed with the feed of the present invention was used as the blank control group, the group infected with Vibrio and not fed with the feed of the present invention was used as the control group, and the groups infected with Vibrio and fed with the feeds of Example 1, Example 2 and Example 3 were respectively the experimental groups 1, 2 and 3, and they were fed for 30 days. 9 shrimps were randomly taken from each experimental group at 0 d, 30 d, the 1st day after infection (P1d), and the 10th day after infection (P10d) to collect hemolymph, and an equal volume of anticoagulant was added and mixed evenly. Then it was centrifuged at 4000 r / min for 10 min at 4 °C, and the supernatant was taken. The total protein, albumin, glucose, triglyceride, total cholesterol contents and the activities of alanine aminotransferase, aspartate aminotransferase and creatine kinase in the hemolymph were detected according to the instructions of the corresponding kits of Nanjing Jiancheng Bioengineering Institute (for details, see Figure 7 , where it should be noted that: a in 7-1 to 7-10 represents the blank control group, b represents the dosage of baicalein flavonoid is 100 mg / kg, c represents the dosage of baicalein flavonoid is 200 mg / kg, d represents the dosage of baicalein flavonoid is 400 mg / kg, and e represents the group only infected).

[0066] It can be found through the experiment that: there were no significant differences in the albumin and total protein contents among the experimental groups. At P1d, except for the blank control group, the albumin and total protein contents in the control group and experimental groups 1-3 were significantly decreased. Compared with the control group, the levels of plasma albumin and total protein reduction in the shrimps of experimental groups 1-3 after Vibrio infection were significantly reduced (P < 0.05), and with the increase of the baicalein flavonoid dose in the experimental groups, the levels of plasma albumin and total protein reduction in the shrimps after bacteria infection gradually decreased. On the 10th day after infection (P10d), the triglyceride and total cholesterol levels of the shrimps in each group gradually recovered (for details, see Figure 7-1 and Figure 7-2 ).

[0067] It can be found through the experiment that after feeding the additive for 30 d, there was little difference in the blood glucose level in the hemolymph of the shrimps among the groups (P > 0.05); on the 1st day after infection (P1d), the blood glucose in the hemolymph of the shrimps in the group only infected increased sharply, and the blood glucose level in the group using the additive was significantly lower than that in the group only infected (P < 0.05), and with the increase of the baicalein flavonoid dose, the increasing amplitude of the blood glucose level in the hemolymph of the shrimps gradually decreased; on the 10th day after infection (P10d), except that the blood glucose level in the group only infected was slightly higher, the blood glucose in the other groups recovered to the normal level (for details, see appendix Figure 7-3 ).

[0068] It can be found through experiments that there is little difference in the triglyceride levels in the hemolymph of prawns among different groups (P>0.05), and the total cholesterol level gradually decreases with the increase of the dose of baicalin flavonoids (P<0.05); on the 1st day after infection (P1d), only in the control group, the triglyceride and total cholesterol levels in the hemolymph of prawns increased sharply. The triglyceride and total cholesterol levels in experimental groups 1-3 were significantly lower than those in the infection-only group, and with the increase of the dose of baicalin flavonoids in the experimental groups, the increasing amplitude of the triglyceride and total cholesterol levels in the hemolymph of prawns gradually decreased; on the 10th day after infection (P10d), the triglyceride and total cholesterol levels of prawns in experimental groups 1-3 gradually recovered (for details, see Figure 7-3 and Figure 7-4 ).

[0069] It can be found through experiments that there are no significant differences in the creatine kinase, glutamic-oxaloacetic transaminase and glutamic-pyruvic transaminase levels in the hemolymph of prawns among different groups (P>0.05); at P1d, only in the control group, the levels of the three enzymes in the hemolymph of prawns increased sharply. The levels of the three enzymes in experimental groups 1-3 were significantly lower than those in the infection-only group, and with the increase of the dose of baicalin flavonoids in the experimental groups, the increasing amplitude of the creatine kinase, glutamic-oxaloacetic transaminase and glutamic-pyruvic transaminase levels in the hemolymph of prawns gradually decreased. At P10d, the creatine kinase, glutamic-oxaloacetic transaminase and glutamic-pyruvic transaminase levels of prawns in experimental groups 1-3 gradually recovered (for details, see Figure 7-6 、7-7 and 7-8).

[0070] At 0d, 30d, P1d and P10d respectively, 3 prawns were randomly selected from each experimental group to collect hemolymph. After adding anticoagulant with the same volume as the hemolymph and mixing evenly, 20% formaldehyde solution was added for fixation to make the final solution contain 10% formaldehyde. The fixed hemolymph solution was used for total hemocyte count (THCs) with a hemocytometer. It can be found through experiments that the THCs of the control group changed little at the 4 sampling time points. After 30 days of cultivation, the THCs of experimental groups 1-3 were significantly higher than those of the control group, and the THCs were positively correlated with the drug dose. On the 1st day after infection, the THCs of the control group and experimental groups 1-3 all decreased significantly (P<0.05); on the 10th day after infection, the THCs of experimental groups 1-3 gradually recovered and increased. The THCs of experimental group 3 were significantly higher than those of the control group and experimental groups 1 and 2 (P<0.05), and the THCs were positively correlated with the drug dose (for details, see Figure 7 -9).

[0071] Three shrimps were randomly selected from each experimental group at 0d, 30d, P1d, and P10d, and hemolymph was collected. An equal volume of anticoagulant was added and mixed. The mixture was centrifuged at 4000r / min for 10min at 4℃. The supernatant was taken and mixed with 900μL double distilled water. The absorbance was measured at 335nm, and the hemocyanin (HEM) content was calculated using the formula: E335nm (mM) = 17.26 × OD335. The experiment showed that after 30 days of culture, the HEM in the experimental groups 1-3 was significantly higher than that in the control group (P<0.05), and the HEM was positively correlated with the drug dose. On the first day after infection, HEM in the control group and experimental groups 1-3 decreased significantly (P<0.05); on the tenth day after infection, HEM in experimental groups 1-3 gradually recovered and increased, and HEM in experimental group 3 was significantly higher than that in the control group and experimental groups 1 and 2 (P<0.05). HEM was positively correlated with drug dose (see Figure 7-1 0).

[0072] In summary, based on the comprehensive analysis of various blood physiological and biochemical indicators, it can be seen that the high-dose group of Scutellaria baicalensis flavonoids maintained the best shrimp hepatopancreatic function, energy metabolism and other body functions.

[0073] Experimental Example 7: Effects of Litopenaeus vannamei on intestinal flora

[0074] During the entire experimental period, Proteobacteria (corresponding to the light blue in the attached figure), Bacteroidetes (corresponding to the red in the attached figure) and Firmicutes (corresponding to the green in the attached figure) are the three main microbial groups in the intestines of shrimp. At the beginning of the experiment, the abundance of Proteobacteria in the intestines of shrimp was the highest, followed by Bacteroidetes and Firmicutes. After 30 days of feeding the additive, the abundance of Proteobacteria in the intestines of shrimp decreased, while the abundance of Bacteroidetes and Firmicutes increased. Among them, the abundance of bacteria in the scutellaria flavonoids group was Bacteroidetes>Firmicutes>Proteobacteria. After infection with Vibrio, the abundance of Proteobacteria in the intestines of shrimp in the low- and medium-dose scutellaria flavonoids groups increased rapidly, becoming the most important intestinal flora. The high-dose scutellaria flavonoids group still maintained the highest abundance of Bacteroidetes in the intestine (49.3%). For details, see Figure 8 From the changes in intestinal flora, the high-dose group of scutellaria flavonoids showed stronger resistance to Vibrio infection in shrimp.

[0075] In summary, it can be found that adding scutellaria baicalensis flavonoids to the diet can promote the growth of Penaeus vannamei, significantly improve the nonspecific immune function and anti-Vibrio infection ability of the shrimp, and maintain the stability of the shrimp's energy generation and intestinal flora. After comparing the experimental effects of different doses of scutellaria baicalensis flavonoids, the recommended dosage for shrimp production is 400 mg / kg.

[0076] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims

1. A Vibrio-resistant and growth-promoting feed for shrimp, characterized in that: The feed comprises the following components: a basic feed and scutellaria baicalensis flavonoids, wherein the scutellaria baicalensis flavonoids are mixed with the basic feed in an addition amount of 100-400 mg per 1 kg of the basic feed.

2. The Vibrio-resistant and growth-promoting feed for shrimp according to claim 1, characterized in that: The scutellaria baicalensis flavonoids are mixed with the basic feed in an amount of 400 mg per 1 kg of the basic feed.

3. The Vibrio-resistant and growth-promoting feed for shrimp according to claim 1, characterized in that: The basic feed comprises the following components by weight: 200g fish meal, 300g wheat gluten, 200g wheat flour, 180g cellulose, 25g fish oil, 25g soybean oil, 20g phospholipids, 20g adhesive, 10g choline chloride, 10g complex vitamins and 10g complex minerals.

4. The Vibrio-resistant and growth-promoting feed for shrimp according to claim 3, characterized in that: The complex vitamins in every 1 kg of basic feed include the following components by weight: 45 mg riboflavin, 25 mg thiamine, 10 mg vitamin K3, 800 mg inositol, 20 mg pyridoxine hydrochloride, 0.1 mg vitamin B12, 60 mg calcium pantothenate, 1.2 mg biotin, 32 mg vitamin A, 5 mg vitamin D, 200 mg niacin, 20 mg folic acid and 120 mg vitamin E.

5. The Vibrio-resistant and growth-promoting feed for shrimp according to claim 3, characterized in that: The complex minerals in every 1kg of basic feed include the following components by weight: KI 0.8mg, NaF 2mg, Fe2(SO4)380mg, ZnSO450mg, CoCl2·6H2O 50mg, CuSO4·5H2O 10mg, MgSO4200mg, Nacl 100mg, Ca(H2PO4)23000mg.

6. The Vibrio-resistant and growth-promoting feed for shrimp according to claim 3, characterized in that: In every 1 kg of basic feed, the crude protein content of the fish meal is 689.9 g / kg dry matter, and the crude fat content is 78.1 g / kg dry matter; the crude protein content of the wheat gluten is 790.5 g / kg dry matter, and the crude fat content is 1.8 g / kg dry matter; the crude protein content of the wheat flour is 165.0 g / kg dry matter, and the crude fat content is 15.8 g / kg dry matter.

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