Application of astaxanthin as feed additive in improving disease resistance of Macrobrachium rosenbergii

Abstract

The invention discloses the application of astaxanthin as a feed additive in improving the disease resistance of Macrobrachium rosenbergii, and belongs to the field of feed additives. The invention improves the survival rate of Macrobrachium rosenbergii juveniles infected with DIV1 by supplementing astaxanthin in feed, and reduces the abundance of pathogenic bacteria in the intestinal tract of Macrobrachium rosenbergii after infection with DIV1, thereby achieving the purpose of preventing the outbreak of Macrobrachium rosenbergii diseases caused by DIV1, and is a sustainable and effective method for preventing and treating Macrobrachium rosenbergii DIV1 diseases.

Description

Technical Field

[0001] The invention relates to the field of feed additives, and in particular to the application of astaxanthin as a feed additive in improving the disease resistance of Macrobrachium rosenbergii. Background Art

[0002] With the rapid increase in aquaculture production, the frequency of disease outbreaks caused by high-density intensive farming is also increasing, causing serious economic losses and posing a challenge to the sustainable development of aquaculture. Although some chemical drugs are often used to prevent and treat outbreaks of aquaculture diseases, long-term use can lead to drug resistance in pathogens. Vaccination is also a strategy to prevent disease outbreaks, but it is ineffective against crustaceans because they do not have the ability to form long-term acquired immunity, and the number of vaccines licensed for marketing in aquaculture is limited. Therefore, research on safe and effective feed additives to improve the disease resistance of aquatic animals is of great significance to achieving sustainable aquaculture.

[0003] Macrobrachium rosenbergii is one of the important freshwater shrimp species in my country. In 2022, the national Macrobrachium rosenbergii seedling production was more than 30 billion, the farming area was about 495,000 mu, and the total farming output was 177,000 tons. While the Macrobrachium rosenbergii industry is developing rapidly, disease problems have also begun to emerge, seriously affecting the economic benefits of the Macrobrachium rosenbergii industry. Among them, the Macrobrachium rosenbergii "white spot disease" caused by Decapoda iridovirus 1 (DIV1) can have a mortality rate of up to 100%. Therefore, it is very necessary to adopt effective and sustainable methods to prevent and control the Macrobrachium rosenbergii DIV1 disease. Summary of the invention

[0004] The purpose of the present invention is to provide an application of astaxanthin as a feed additive in improving the disease resistance of Macrobrachium rosenbergii, so as to solve the problems existing in the above-mentioned prior art. The present invention improves the survival rate of Macrobrachium rosenbergii juveniles after infection with DIV1 by supplementing astaxanthin in the feed, and at the same time reduces the abundance of pathogenic bacteria in the intestine of Macrobrachium rosenbergii after infection with DIV1, thereby achieving the purpose of preventing the outbreak of Macrobrachium rosenbergii diseases caused by DIV1.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides application of astaxanthin in preparing a feed additive for improving the disease resistance of Macrobrachium rosenbergii to decapod iridovirus 1.

[0007] Optionally, astaxanthin improves the disease resistance of Macrobrachium rosenbergii to Decapoda iridovirus 1 by increasing the survival rate of Macrobrachium rosenbergii after infection with Decapoda iridovirus 1.

[0008] Optionally, astaxanthin improves the disease resistance of Macrobrachium rosenbergii to Decapoda iridovirus 1 by reducing the abundance of intestinal pathogenic bacteria after Macrobrachium rosenbergii is infected with Decapoda iridovirus 1.

[0009] Optionally, the intestinal pathogenic bacteria include Lactococcus garvieae and Acinetobacter junii.

[0010] The present invention also provides the use of astaxanthin as a feed additive in preparing a feed for improving the disease resistance of Macrobrachium rosenbergii to decapod iridovirus 1.

[0011] Optionally, in the feed, the mass fraction of astaxanthin is 0.1%~1%.

[0012] Further optionally, in the feed, the mass fraction of astaxanthin is 0.5%.

[0013] The present invention also provides a breeding method for improving the disease resistance of Macrobrachium rosenbergii to Decapoda iridovirus 1, comprising the steps of adding astaxanthin to the feed of Macrobrachium rosenbergii and feeding the Macrobrachium rosenbergii with the feed added with astaxanthin.

[0014] Optionally, in the astaxanthin-added feed, the mass fraction of astaxanthin is 0.1%~1%.

[0015] Further optionally, in the astaxanthin-added feed, the mass fraction of astaxanthin is 0.5%.

[0016] The present invention discloses the following technical effects:

[0017] The present invention improves the survival rate of Macrobrachium rosenbergii juveniles infected with DIV1 by supplementing astaxanthin in feed, and reduces the abundance of pathogenic bacteria in the intestine of Macrobrachium rosenbergii after infection with DIV1, thereby achieving the purpose of preventing the outbreak of Macrobrachium rosenbergii diseases caused by DIV1. The present invention is a sustainable and effective method for preventing and controlling Macrobrachium rosenbergii DIV1 diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0019] Figure 1 This is a correlation analysis chart between liver astaxanthin content and feed astaxanthin content;

[0020] Figure 2This is the cumulative survival rate of different groups of Macrobrachium rosenbergii after infection with DIV1, where * indicates P < 0.05;

[0021] Figure 3 Abundance diagram of intestinal pathogenic bacteria Lactococcus_garvieae (A) and Acinetobacter_junii (B) in the control group and A2 treatment group after infection with Macrobrachium rosenbergii at DIV1; ** indicates P < 0.01. DETAILED DESCRIPTION

[0022] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0023] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0024] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0025] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0026] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0027] The present invention improves the survival rate of Macrobrachium rosenbergii juveniles infected with DIV1 by supplementing astaxanthin in feed, reduces the abundance of intestinal pathogenic bacteria after Macrobrachium rosenbergii is infected with DIV1, and prevents the outbreak of Macrobrachium rosenbergii diseases caused by DIV1.

[0028] In a specific embodiment, the mass fraction of astaxanthin in the feed is 0.1%~1%.

[0029] Optionally, the mass fraction of astaxanthin in the feed is 0.5%.

[0030] In a specific embodiment, the feed is prepared with fish meal, soybean meal, chicken meal and soybean protein concentrate as protein sources, and fish oil and soybean oil in a mass ratio of 1:1 as fat sources.

[0031] Optionally, the crude protein content of the above feed is 40.38% ~ 41.27%, the fat content is 8.9% ~ 9.1%, the ash content is 11.8% ~ 12.3%, and the moisture content is 6.4% ~ 8.5%.

[0032] The technical solution of the present application is described in detail below in conjunction with specific embodiments.

[0033] Unless otherwise specified, the percentages involved in the embodiments of the present invention are all understood to be mass percentages, the experimental methods are all conventional methods in the art, and the materials or reagents used can be purchased through conventional channels.

[0034] Example

[0035] 1. Experimental Methods

[0036] 1. Feed formula

[0037] Four kinds of feeds were prepared with fish meal, soybean meal, chicken meal and soybean protein concentrate as protein sources, and fish oil and soybean oil with a mass ratio of 1:1 as fat sources, including control feed without astaxanthin (A0), feed with astaxanthin added at 0.1% (A1), 0.5% (A2) and 1% (A3). The feed formulas are shown in Table 1. The nutritional components of the four groups of feeds are shown in Table 2. The crude protein content was 40.38%~41.27%, the fat content was 8.9%~9.1%, the ash content was 11.8%~12.3%, the moisture content was 6.4%~8.5%, the actual astaxanthin content was 0.036mg / kg, 107mg / kg, 521mg / kg and 955mg / kg, respectively, and the total carotenoid content was 0.2mg / 100g, 4.4mg / 100g, 20.3mg / 100g and 34.4mg / 100g, respectively. The feed raw materials were passed through a 40-mesh sieve, mixed thoroughly, and made into 2 mm particles using a feed machine (SYSLG30-IV), naturally air-dried, and sealed and stored in a 4°C refrigerator for later use.

[0038] Table 1 Feed formula (% dry matter)

[0039]

[0040] Table 2 Nutritional composition of feed (% dry matter) and content of astaxanthin and carotenoids

[0041]

[0042] 2. Experimental shrimp and feeding management

[0043] Macrobrachium rosenbergii juveniles were temporarily cultured in a square pond with an area of ​​8 m2 × 1.1 m × 1.5 m for 3 weeks. One day before the experiment, 600 shrimps of uniform size (0.22 ± 0.019 g) were randomly selected and evenly distributed to 12 independent 300-liter (0.97 m × 0.57 m × 0.61 m) water tanks. A total of 4 treatment feeds (Table 1) were set up in 3 parallel water tanks for each feed group, with 50 shrimps in each tank. The culture period was 8 weeks. Feeding was done twice a day, and feeding was done at apparent satiation. During the culture period, the water temperature was 23-27 °C, the pH was 7.4-8.3, the dissolved oxygen was greater than 7.0 mg / L, and the ammonia nitrogen was less than 0.05 mg / L.

[0044] 3. DIV1 virus infection experiment

[0045] Preparation of DIV1 crude virus extract: positive Macrobrachium rosenbergii muscle and PBS buffer were placed in a sterile enzyme-free glass grinding tube at a ratio of 1:10. The grinding tube was placed in an ice water bath and slowly ground into a homogenate. The homogenate was then transferred to a centrifuge tube and centrifuged at 3000 r / min at 4°C for 20 min in a low-temperature centrifuge. The supernatant was taken and centrifuged again at 8000 r / min at 4°C for 25 min. The supernatant was taken again and filtered through a 0.22μm pore size filter membrane. The prepared crude virus extract was stored at 4°C.

[0046] Infection experiment: After the breeding experiment, the number and total weight of shrimps in each water tank were counted, and liver samples of 2 shrimps were randomly selected from each water tank for astaxanthin detection. Then 21 shrimps (7 shrimps from each water tank) were randomly selected from the experimental shrimps cultured with each treatment feed for DIV1 challenge test. The challenge test temperature was 25°C. According to the preliminary experiment, the median lethal concentration of DIV1 infection for 96 hours was 1.95×10 7 Each shrimp was injected with 50 μL of the semi-lethal concentration of the crude virus extract. 24 hours after the challenge, the intestinal microorganisms of 6 shrimps in each treatment group were selected for pathogen abundance analysis, and the remaining shrimps were used to calculate the cumulative survival rate after the challenge, which was calculated every 24 hours for 144 hours.

[0047] 4. Sample and data analysis

[0048] The detection of liver astaxanthin was based on the method of SN / T 2327-2009; Shanghai Meiji Biomedical Technology Co., Ltd. was commissioned to sequence the 16S rRNA of intestinal microorganisms using the Illumina Miseq PE300 / NovaSeq PE250 platform; the Kaplan-Meier method was used to estimate the cumulative survival rate of experimental shrimp in different treatment groups after infection with DIV1, and the log-rank method was used to test the difference in cumulative survival rate. SPSS 19.0 was used for the homogeneity of variance test, one-way ANOVA, Duncan's test, and linear or multiple regression analysis, and P < 0.05 was considered significant.

[0049] 2. Experimental Results

[0050] 1. Growth of Macrobrachium rosenbergii

[0051] When juvenile Macrobrachium rosenbergii was cultured on feeds supplemented with astaxanthin (A1~A3) for 8 weeks, there was no significant difference in final weight, weight gain rate and specific growth rate compared with the control group on feeds without astaxanthin (A0), indicating that astaxanthin feed does not affect the growth of juvenile Macrobrachium rosenbergii (Table 3).

[0052] Table 3 Effects of different treatment groups of feed on the growth of juvenile Macrobrachium rosenbergii

[0053]

[0054] 2. Liver astaxanthin content

[0055] The astaxanthin content in the liver of juvenile Macrobrachium rosenbergii increased significantly with the increase of astaxanthin content in the feed. The astaxanthin content in the liver of groups A1, A2 and A3 increased significantly by 4.99, 8.21 and 13.74 times compared with the control group A0, respectively (Table 4). Figure 1 As shown in the figure, the astaxanthin content in liver was significantly linearly correlated with the astaxanthin content in feed (P < 0.05), and the regression equation was Y = 0.05179 * X + 10.83 (Y: astaxanthin content in liver, X: astaxanthin content in feed, R 2 =0.93).

[0056] Table 4 Effects of different treatment groups of feed on astaxanthin content in the liver of Macrobrachium rosenbergii

[0057]

[0058] 3. Cumulative survival rate of DIV1 virus infection

[0059] Feeding the feed supplemented with astaxanthin can improve the cumulative survival rate of Macrobrachium rosenbergii juveniles after infection with DIV1, and the A2 group was significantly higher than the control group ( Figure 2, P<0.05). As shown in Table 5, the cumulative survival rate of the control group after 144 hours of infection was 40%, and the cumulative survival rates of the A1, A2 and A3 astaxanthin-treated groups were 67%, 90% and 77%, respectively, which were 27%, 50% and 37% higher than those of the control group.

[0060] Table 5 Cumulative survival rate of Macrobrachium rosenbergii 144 hours after infection with DIV1

[0061]

[0062] 4. Abundance of intestinal pathogens after DIV1 infection

[0063] Lactococcus garvieae and Acinetobacter junii are the main pathogens of aquatic animals. The results of the present embodiment show that after Macrobrachium rosenbergii infects DIV1, the abundance of intestinal pathogenic bacteria Lactococcus garvieae and Acinetobacter junii in the control group is 27.21% and 2.58% respectively. As shown in Table 6, compared to the control group, the abundance of Lactococcus garvieae and Acinetobacter junii in A1 group is reduced by 19.69% and 1.93% respectively, the abundance of Lactococcus garvieae and Acinetobacter junii in A2 group is reduced by 24.51% and 2.35% respectively, and the abundance of Lactococcus garvieae and Acinetobacter junii in A3 group is reduced by 13.13% and 2.27% respectively. Among them, the abundance of Lactococcus_garvieae and Acinetobacter_junii in group A2 was significantly lower than that in the control group ( Figure 3 , P<0.01).

[0064] Table 6 Abundance of intestinal pathogenic bacteria Lactococcus_garvieae and Acinetobacter_junii in different treatment groups after infection with DIV1

[0065]

[0066] The above examples show that adding 107 mg / kg, 521 mg / kg and 955 mg / kg of astaxanthin to feed has no effect on the growth of Macrobrachium rosenbergii juveniles, but can significantly increase the astaxanthin content in the liver of Macrobrachium rosenbergii juveniles, increase the cumulative survival rate of Macrobrachium rosenbergii juveniles after infection with DIV1, and reduce the abundance of intestinal pathogens Lactococcus_garvieae and Acinetobacter_junii after Macrobrachium rosenbergii is infected with the virus. At the same time, the study found that the astaxanthin content in the liver of Macrobrachium rosenbergii and the astaxanthin content in the feed were significantly linearly correlated (Y=0.05179*X+10.83, Y: liver astaxanthin content, X: feed astaxanthin content, R 2 =0.93). Adding 521 mg / kg astaxanthin to feed had the best effect.

[0067] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. Application of astaxanthin in the preparation of feed additives for improving the disease resistance of Macrobrachium rosenbergii to decapod iridovirus 1.

2. The use according to claim 1, characterized in that: Astaxanthin improves the disease resistance of Macrobrachium rosenbergii to Decapoda iridovirus-1 by increasing the survival rate of Macrobrachium rosenbergii after infection with Decapoda iridovirus-1.

3. The use according to claim 1, characterized in that: Astaxanthin improves the disease resistance of Macrobrachium rosenbergii to Decapoda iridovirus 1 by reducing the abundance of intestinal pathogenic bacteria after Macrobrachium rosenbergii is infected with Decapoda iridovirus 1.

4. The use according to claim 3, characterized in that: The intestinal pathogens include Lactococcus garvae ( Lactococcus_garvieae ) and Acinetobacter junii ( Acinetobacter_junii ).

5. The application of astaxanthin as a feed additive in the preparation of feed for improving the disease resistance of Macrobrachium rosenbergii to decapod iridovirus 1.

6. The use according to claim 5, characterized in that: In the feed, the mass fraction of astaxanthin is 0.1%~1%.

7. The use according to claim 5, characterized in that: In the feed, the mass fraction of astaxanthin is 0.5%.

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