Method and system for testing influence of natural astaxanthin on growth, body color and immunity of macrobrachium rosenbergii

Abstract

The invention belongs to the technical field of biology, and discloses a method and a system for testing influence of natural astaxanthin on growth, body color and immunity of macrobrachium rosenbergii. Natural astaxanthin with different addition amounts is fed, and the influence of natural astaxanthin on growth, immunity, antioxidant enzyme activity and related gene expression of macrobrachium rosenbergii is detected and evaluated; reference can be provided for utilization of astaxanthin in healthy culture of macrobrachium rosenbergii. The addition amount of the astaxanthin is 100 mg kg <-1 > and 200 mg kg <-1 >, so that the growth performance and the body color of the macrobrachium rosenbergii can be remarkably improved, and the content of the astaxanthin in muscles is increased. Meanwhile, the astaxanthin can improve the immunity and antioxidant capacity of the macrobrachium rosenbergii. However, factors influencing the absorption of the astaxanthin in the macrobrachium rosenbergii still need to be further researched. The research result provides reference data for application of astaxanthin in crustacean feed and promotion of healthy development of aquaculture industry.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a method and a system for testing the influence of natural astaxanthin on the growth, body color and immunity of Macrobrachium rosenbergii. Background Art

[0002] Astaxanthin (Ax), also known as astaxanthin and astaxanthin, is a carotenoid extracted from animals such as shrimp and crab (Yuan et al., 2014). Astaxanthin mainly includes natural astaxanthin and synthetic astaxanthin. The former is extracted from algae, yeast, bacteria and other organisms (Asker et al., 2018; Fang et al., 2019; Leytonet al., 2019; Khadilkar et al., 2023), while the latter is obtained by chemical synthesis (Xian et al., 2019). Although many aquatic animals are rich in astaxanthin, they cannot synthesize it from scratch, but are absorbed from food and then enriched in the body (Yaakob et al., 2014; Yu and Liu, 2020). Astaxanthin, a high-value ketocarotenoid that is not easily soluble in water, is a strong antioxidant in nature (Zhao et al., 2019; Zhang et al., 2023). It mainly functions by capturing free radicals on the surface and inside of phospholipid membranes to achieve its efficient antioxidant function (Goto et al., 2001; Paixao et al., 2011). Astaxanthin has good antioxidant activity, it is often used to improve the body's immunity, disease resistance and anti-stress ability, so as to better resist various environmental stresses (Chien and Shiau, 2005; Wang et al., 2018; Elia et al., 2019). For example, when 30 mg kg-1 of astaxanthin was added to the feed of rainbow trout (Oncorhynchus mykiss) for 4 months, its weight gain rate increased by 20.75% and the activity of glutathione peroxidase increased significantly, proving that astaxanthin can improve the growth performance of rainbow trout and improve the body's anti-stress ability (Long et al., 2023); adding 0.6% astaxanthin to the feed of Procambarus clarkii is beneficial to the stability of the intestinal microbial flora of Procambarus clarkii and alleviate its immune damage (Cai et al., 2022). Similarly, adding astaxanthin to the feed of Litopenarus vannamei not only improved its growth performance, but also enhanced its immunity and antioxidant capacity (Zhao et al., 2022).

[0003] In addition to the above functions, astaxanthin also has good coloring function (Fakhri et al., 2018; Jiang et al., 2022; Debnath et al., 2024). For example, in rainbow trout farming, as the astaxanthin content increases, the redness (a* value) of rainbow trout fillets will gradually become darker (Long et al., 2023). Similarly, in the breeding of ornamental fish species, astaxanthin is often added to feed to change the body color of various ornamental fish. For example, adding natural astaxanthin to the feed of Symphysodon haraldi (Wang et al., 2016) and koi (Cyprinus carpio L.) (Wang et al., 2019) can significantly increase the redness (a*) and yellowness (b*) of both, thereby achieving a beautiful color effect. In addition, astaxanthin can not only change the astaxanthin content in muscle, but also shorten its molting cycle. For example, in crustaceans, after the white shrimp Exopalaemon carinicauda was fed a feed containing astaxanthin for 56 days, the body surface of the shrimp became noticeably redder and the contents of total astaxanthin and free astaxanthin in the abdominal muscle increased (Zhang et al., 2021); when 80 mg kg-1 of astaxanthin was added to the feed of juvenile Penaeus vannamei, the molting cycle of the juvenile shrimp was significantly shortened (Flores et al., 2007).

[0004] Macrobrachium rosenbergii is a freshwater aquaculture species that is easy to raise, grows fast, has delicious meat and high nutritional value. According to the China Fisheries Statistical Yearbook, the annual production of Macrobrachium rosenbergii in China exceeded 190,000 tons in 2023 (2024). Macrobrachium rosenbergii farming not only creates income and increases efficiency for farmers, but also promotes the development of China's agricultural economy. At present, the only research on astaxanthin in Macrobrachium rosenbergii is: Jin Zhengyu et al. added astaxanthin to Macrobrachium rosenbergii feed, and the results showed that astaxanthin can promote the increase of carotenoid content in its body (Jin et al., 1999); recently, Xu et al. found that feeding Macrobrachium rosenbergii with feed containing astaxanthin not only enhanced its growth and immune performance (2022), but also promoted the metamorphosis of Macrobrachium rosenbergii (2024). However, research on other aspects of astaxanthin in Macrobrachium rosenbergii is still relatively weak.

[0005] Through the above analysis, the problems and defects of the prior art are as follows:

[0006] Research on other aspects of astaxanthin's effects on Macrobrachium rosenbergii is still relatively weak. Summary of the invention

[0007] In view of the problems existing in the prior art, the present invention provides a method for testing the effects of natural astaxanthin on the growth, body color and immunity of Macrobrachium rosenbergii.

[0008] The present invention is achieved in that a method for testing the effect of natural astaxanthin on the growth, body color and immunity of Macrobrachium rosenbergii comprises:

[0009] Step 1, sample collection;

[0010] Step 2, growth index calculation;

[0011] Step 3, body color determination; immune and antioxidant enzyme activity determination; immune and molting gene expression level determination; astaxanthin content determination;

[0012] Step 4: Statistical analysis of data.

[0013] Further, the sample collection:

[0014] Sample collection was carried out in accordance with the animal collection rules of the Animal Experiment Ethics Committee of the Pearl River Fisheries Research Institute; fasting for 24 hours before each sampling; sampling was performed in the 10th and 20th weeks of breeding; the number of surviving Macrobrachium rosenbergii in each group was counted and the survival rate was calculated; 20 shrimps were randomly collected from each group to count the weight and body length; 12 live shrimps were randomly taken from each group for live shrimp body color determination, and in the 20th week, 8 shrimps were taken from each group and boiled in water for 3 minutes to determine the body color of cooked shrimps; 12 Macrobrachium rosenbergii were taken from each group; tissues of every 3 shrimps were mixed into 1 portion, for a total of 4 portions, the same below; hemolymph was extracted along the base of the fifth stepping foot with a 1mL disposable syringe containing an anticoagulant, centrifuged at 1000xg for 5 minutes, and the supernatant was aspirated and stored at -80℃; the hepatopancreas was taken and weighed for calculation of the liver-to-body ratio and evaluation of subsequent related enzyme activities and expression levels of immune-related genes; muscles from the 20th week were taken for detection of astaxanthin content;

[0015] According to the structural changes in the distal region of the fifth abdominal foot, the molting process of Macrobrachium rosenbergii in each group was divided into three periods: intermolting, premolting and postmolting. Four hepatopancreas samples were collected during each molting period; 3 samples / sample were used to evaluate the expression levels of related genes in different treatment groups at different molting periods.

[0016] All hepatopancreas and muscle tissues were frozen with liquid nitrogen and then stored at -80°C for subsequent determination of various indicators.

[0017] Further, the growth index is calculated as:

[0018] At the 10th and 20th weeks, the body length, body weight and hepatopancreas weight of Macrobrachium rosenbergii were measured using tpsDig2 software (F. James Rohlf, Stony Brook University, Stony Brook, NY, USA) and a Mettler Toledo AL-204 precision balance (Mettler Toledo, Inc., Shanghai, China). The following formulas were used to calculate growth index parameters such as survival rate, weight gain rate, specific growth rate and liver-to-body ratio:

[0019] Survival rate (SR,%) = N t / N0×100

[0020] Weight gain rate(WGR,%)=(W t -W0) / W0×100

[0021] Specific growth rate (SGR,% / d)=(lnW t -lnW0) / t

[0022] Hepatosomatic index(HSI,%)=W h / W b ×100

[0023] Where: N0 is the initial number (tail); N t is the final weight (tail); W0 is the initial weight (g); W t is the final body weight (g); t is the number of culture days (d); W h W is the weight of hepatopancreas (g); b is body weight (g).

[0024] Further, the body color determination:

[0025] At the 10th and 20th weeks of culture, the L of Macrobrachium rosenbergii was measured using a 3nh colorimeter (Shenzhen Sanlian Technology Co., Ltd., Shenzhen, China). * value (represents brightness, the larger the value, the lighter the color), a * Value(+a * Reddish, -a * Greenish) and b * Value(+b * Yellowish, -b * The measured parts were the 1st, 3rd and 5th abdominal segments (Zhang et al., 2021). In addition, at the 20th week of culture, 8 Macrobrachium rosenbergii were taken from each group and boiled in boiling water for 3 minutes, then removed and cooled at room temperature to measure the color value of the cooked shrimp. The color difference value ΔE was calculated using the following formula:

[0026]

[0027] Where: L * is the brightness of the treatment group; is the brightness of the control group; a * is the redness of the treatment group, b is the redness of the control group; * is the yellowness of the treatment group, Yellowness of the control group.

[0028] Further, the immune and antioxidant enzyme activities were determined:

[0029] The activities of superoxide dismutase (SOD), catalase (CAT), malondialdehyde (MDA), alkaline phosphatase (AKP) and lysozyme (LZM) in the hepatopancreas and hemolymph of Macrobrachium rosenbergii at the end of the 10th and 20th weeks were determined by WST-1 method, ammonium molybdate method, thiobarbital acid (TBA) reaction method, microenzyme labeling method and turbidimetry, respectively, to evaluate the effects of astaxanthin on immunity and antioxidant. The operation steps were in accordance with the instructions of the kit of Nanjing Jiancheng Bioengineering Institute (Nanjing, China);

[0030] Determination of immune and molting gene expression levels:

[0031] Total RNA from hepatopancreas was extracted by Trizol method, and then the integrity of RNA was evaluated by 1% agarose gel electrophoresis, and the concentration of RNA was detected by Nanodrop-2000 spectrophotometer. cDNA was synthesized according to the instructions of M-MLV reverse transcription kit. Real-time fluorescence quantitative PCR was performed using StepOnePlus Real-Tim PCR system to evaluate the expression levels of immune and molting-related genes in Macrobrachium rosenbergii after the addition of astaxanthin. The reaction system included 1 μL (50 ng / μL) cDNA, 10 μL 2×SYBRGreen Premix (Universal), 1 μL primer (10 pmol / μL) and 7 μL double distilled water, with a total system of 20 μL. The reaction procedure was as follows: pre-denaturation at 95°C for 30 min; 35 cycles of denaturation at 95°C for 40 s, annealing at 60°C for 45 s, and extension at 72°C for 10 min; data acquisition at 72°C for 10 min, and then melting curves were obtained at 95°C for 5 s, 60°C for 30 s, and 95°C for 15 s.

[0032] Astaxanthin content determination:

[0033] 5 g of muscle sample was extracted with acetonitrile, defatted and concentrated with n-hexane, and the astaxanthin content in the muscle was detected by high performance liquid chromatography.

[0034] Further, the data statistical analysis:

[0035] GraphPad Prism 9 software was used to analyze the differences in growth indicators, chromaticity values, astaxanthin content, immune and antioxidant and related gene expression level parameters of each group; data were expressed as "mean ± standard error", P < 0.05 indicated a significant difference, and P < 0.01 indicated an extremely significant difference.

[0036] Another object of the present invention is to provide a detection system for the effect of natural astaxanthin on the growth, body color and immunity of Macrobrachium rosenbergii, comprising:

[0037] A collection module, used for collecting samples;

[0038] A calculation module, used for calculating growth indicators;

[0039] The measurement module is used for body color measurement, immune and antioxidant enzyme activity measurement, immune and molting gene expression level measurement, and astaxanthin content measurement.

[0040] The analysis module is used to perform differential analysis on growth indexes, chromaticity values, astaxanthin content, immunity and antioxidant and related gene expression level parameters of each group using GraphPadPrism 9 software.

[0041] Another object of the present invention is to provide a computer device, comprising a memory and a processor, wherein the memory stores a computer program, and when the computer program is executed by the processor, the processor executes the steps of the method for testing the effect of natural astaxanthin on the growth, body color and immunity of Macrobrachium rosenbergii.

[0042] Another object of the present invention is to provide a computer-readable storage medium storing a computer program, which, when executed by a processor, enables the processor to execute the steps of the method for testing the effect of natural astaxanthin on the growth, body color and immunity of Macrobrachium rosenbergii.

[0043] Another object of the present invention is to provide an information data processing terminal, which is used to implement a detection system for the effect of natural astaxanthin on the growth, body color and immunity of Macrobrachium rosenbergii.

[0044] In combination with the above technical solutions and the technical problems solved, please analyze the advantages and positive effects of the technical solutions to be protected by the present invention from the following aspects:

[0045] First, the present invention systematically studies the comprehensive effects of natural astaxanthin on the growth, body color, immunity, antioxidant enzyme activity and gene expression level of Macrobrachium rosenbergii, combines the visualized body color determination method, efficient immune enzyme activity and gene expression analysis technology, and accurate astaxanthin content detection, and comprehensively evaluates the multiple biological effects of astaxanthin in Macrobrachium rosenbergii farming. This method helps to optimize the use of feed additives in shrimp farming and improve farming efficiency and product quality through innovative experimental design and data analysis.

[0046] The present invention feeds different amounts of natural astaxanthin to detect and evaluate its effects on the growth, immunity, antioxidant enzyme activity and related gene expression of Macrobrachium rosenbergii, in order to provide a reference for the use of astaxanthin in the healthy cultivation of Macrobrachium rosenbergii.

[0047] Astaxanthin was added at 100 and 200 mg kg -1 It can significantly improve the growth performance and body color of Macrobrachium rosenbergii and increase the astaxanthin content in muscle. At the same time, astaxanthin can improve the immunity and antioxidant capacity of Macrobrachium rosenbergii. However, the factors affecting the absorption of astaxanthin by Macrobrachium rosenbergii need further study. The results of this study provide reference data for the application of astaxanthin in crustacean feed and the promotion of the healthy development of aquaculture.

[0048] Second, the technical solution of the present invention solves several key problems in the prior art in industrial applications and has achieved significant technical progress, which is mainly reflected in the following aspects:

[0049] 1. Comprehensive evaluation method for the effects of natural astaxanthin on the growth, body color and immunity of Macrobrachium rosenbergii

[0050] The existing technology is relatively one-sided in studying the effects of astaxanthin on growth and body color during shrimp farming, and lacks a standardized evaluation process. The present invention provides a complete and scientific evaluation method through a systematic experimental design, including sample collection, body color measurement, growth index calculation, immune and antioxidant enzyme activity detection, molting and gene expression level measurement and other data, which can comprehensively and accurately analyze the multi-dimensional effects of natural astaxanthin on Macrobrachium rosenbergii. This comprehensive evaluation solves the problem of single evaluation methods in the existing technology and provides a scientific basis for the application of natural astaxanthin in shrimp farming.

[0051] 2. Accurate growth index calculation and body color measurement

[0052] In traditional shrimp breeding research, there are subjectivity and errors in the evaluation of growth rate and body color change. The present invention introduces a standardized calculation formula and the use of a 3nh colorimeter to accurately measure shrimp growth indicators such as survival rate, weight gain rate, specific growth rate, etc., and accurately evaluate body color changes by calculating the color difference value ΔE. This standardized growth and body color evaluation method greatly improves the reliability and repeatability of the research results, and solves the problems of inaccurate evaluation methods and large subjective errors in the prior art.

[0053] 3. Systematic detection of immune and antioxidant enzyme activities

[0054] The present invention combines enzyme activity detection (such as SOD, CAT, AKP, etc.) and gene expression analysis technology to evaluate in detail the effects of astaxanthin on the immune system and antioxidant capacity of Macrobrachium rosenbergii. Through quantitative PCR analysis of immune-related genes, a study of the molecular mechanism of astaxanthin in the immune and molting processes is provided, further revealing its potential health-enhancing effect in shrimp farming. This multi-level immune system evaluation method solves the limitation of the prior art that the research on shrimp immunity and antioxidant capacity is not in-depth enough.

[0055] 4. Significant Advances in Data Analysis

[0056] The present invention introduces GraphPad Prism 9 software for data statistical analysis, and performs difference significance analysis on changes in growth, body color, astaxanthin content, immune and antioxidant enzyme activities and gene expression levels. The difference significance is judged by P value, so that the research results have higher statistical significance and explanatory power. Compared with the simple descriptive analysis in the prior art, the statistical method of the present invention significantly improves the scientificity and rigor of data analysis.

[0057] In summary, the present invention solves the problem of lack of comprehensive, accurate and scientific methods in the prior art for evaluating the effects of natural astaxanthin on the growth, body color and immunity of Macrobrachium rosenbergii through standardized and systematic experimental methods, and achieves significant technological progress, promoting industrial applications in the field of shrimp farming and nutritional science. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 It is a flow chart of a method for testing the effect of natural astaxanthin on the growth, body color and immunity of Macrobrachium rosenbergii provided by an embodiment of the present invention.

[0059] Figure 2 It is a system structure block diagram of a method for testing the effect of natural astaxanthin on the growth, body color and immunity of Macrobrachium rosenbergii provided by an embodiment of the present invention. DETAILED DESCRIPTION

[0060] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the 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.

[0061] like Figure 1 As shown, a method for testing the effect of natural astaxanthin on the growth, body color and immunity of Macrobrachium rosenbergii provided in an embodiment of the present invention comprises the following steps:

[0062] S101, sample collection;

[0063] S102, growth index calculation;

[0064] S103, body color determination; immune and antioxidant enzyme activity determination; immune and molting gene expression level determination; astaxanthin content determination;

[0065] S104, data statistical analysis.

[0066] The sample collection provided by the embodiment of the present invention is as follows:

[0067] Sample collection was carried out in accordance with the animal collection rules of the Animal Experiment Ethics Committee of the Pearl River Fisheries Research Institute; fasting for 24 hours before each sampling; sampling was performed in the 10th and 20th weeks of breeding; the number of surviving Macrobrachium rosenbergii in each group was counted and the survival rate was calculated; 20 shrimps were randomly collected from each group to count the weight and body length; 12 live shrimps were randomly taken from each group for live shrimp body color determination, and in the 20th week, 8 shrimps were taken from each group and boiled in water for 3 minutes to determine the body color of cooked shrimps; 12 Macrobrachium rosenbergii were taken from each group; tissues of every 3 shrimps were mixed into 1 portion, for a total of 4 portions, the same below; hemolymph was extracted along the base of the fifth stepping foot with a 1mL disposable syringe containing an anticoagulant, centrifuged at 1000xg for 5 minutes, and the supernatant was aspirated and stored at -80℃; the hepatopancreas was taken and weighed for calculation of the liver-to-body ratio and evaluation of subsequent related enzyme activities and expression levels of immune-related genes; muscles from the 20th week were taken for detection of astaxanthin content;

[0068] According to the structural changes in the distal region of the fifth abdominal foot, the molting process of Macrobrachium rosenbergii in each group was divided into three periods: intermolting, premolting and postmolting. Four hepatopancreas samples were collected during each molting period; 3 samples / sample were used to evaluate the expression levels of related genes in different treatment groups at different molting periods.

[0069] All hepatopancreas and muscle tissues were frozen with liquid nitrogen and then stored at -80°C for subsequent determination of various indicators.

[0070] The growth index calculation provided by the embodiment of the present invention is:

[0071] At the end of the 10th and 20th weeks, the body length, body weight and hepatopancreas weight of Macrobrachium rosenbergii were measured using tpsDig2 software (F. James Rohlf, Stony Brook University, Stony Brook, NY, USA) and a Mettler Toledo AL-204 precision balance (Mettler Toledo, Inc., Shanghai, China). The following formulas were used to calculate growth index parameters such as survival rate, weight gain rate, specific growth rate and liver-to-body ratio:

[0072] Survival rate (SR,%) = N t / N0×100

[0073] Weight gain rate(WGR,%)=(W t -W0) / W0×100

[0074] Specific growth rate (SGR,% / d)=(lnW t -lnW0) / t

[0075] Hepatosomatic index(HSI,%)=W h / W b ×100

[0076] Where: N0 is the initial number (tail); N t is the final weight (tail); W0 is the initial weight (g); W t is the final body weight (g); t is the number of culture days (d); W h W is the weight of hepatopancreas (g); b is body weight (g).

[0077] The body color measurement provided by the embodiment of the present invention is:

[0078] At the 10th and 20th weeks of culture, the L of Macrobrachium rosenbergii was measured using a 3nh colorimeter (Shenzhen Sanlian Technology Co., Ltd., Shenzhen, China). * value (represents brightness, the larger the value, the lighter the color), a * Value(+a * Reddish, -a * Greenish) and b * Value(+b * Yellowish, -b * The measured parts were the 1st, 3rd and 5th abdominal segments (Zhang et al., 2021). In addition, at the 20th week of culture, 8 Macrobrachium rosenbergii were taken from each group and boiled in boiling water for 3 minutes, then removed and cooled at room temperature to measure the color value of the cooked shrimp. The color difference value ΔE was calculated using the following formula:

[0079]

[0080] Where: L * is the brightness of the treatment group; is the brightness of the control group; a * is the redness of the treatment group, b is the redness of the control group; * is the yellowness of the treatment group, Yellowness of the control group.

[0081] The immune and antioxidant enzyme activity assays provided by the embodiments of the present invention are:

[0082] The activities of superoxide dismutase (SOD), catalase (CAT), malondialdehyde (MDA), alkaline phosphatase (AKP) and lysozyme (LZM) in the hepatopancreas and hemolymph of Macrobrachium rosenbergii at the end of the 10th and 20th weeks were determined by WST-1 method, ammonium molybdate method, thiobarbital acid (TBA) reaction method, microenzyme labeling method and turbidimetry, respectively, to evaluate the effects of astaxanthin on immunity and antioxidant. The operation steps were in accordance with the instructions of the kit of Nanjing Jiancheng Bioengineering Institute (Nanjing, China);

[0083] Determination of immune and molting gene expression levels:

[0084] Total RNA from hepatopancreas was extracted by Trizol method, and then the integrity of RNA was evaluated by 1% agarose gel electrophoresis, and the concentration of RNA was detected by Nanodrop-2000 spectrophotometer. cDNA was synthesized according to the instructions of M-MLV reverse transcription kit. Real-time fluorescence quantitative PCR was performed using StepOnePlus Real-Tim PCR system to evaluate the expression levels of immune and molting-related genes in Macrobrachium rosenbergii after the addition of astaxanthin. The reaction system included 1 μL (50 ng / μL) cDNA, 10 μL 2×SYBRGreen Premix (Universal), 1 μL primer (10 pmol / μL) and 7 μL double distilled water, with a total system of 20 μL. The reaction procedure was as follows: pre-denaturation at 95°C for 30 min; 35 cycles of denaturation at 95°C for 40 s, annealing at 60°C for 45 s, and extension at 72°C for 10 min; data acquisition at 72°C for 10 min, and then melting curves were obtained at 95°C for 5 s, 60°C for 30 s, and 95°C for 15 s.

[0085] Astaxanthin content determination:

[0086] 5 g of muscle sample was extracted with acetonitrile, defatted and concentrated with n-hexane, and the astaxanthin content in the muscle was detected by high performance liquid chromatography.

[0087] The data statistical analysis provided by the embodiment of the present invention is as follows:

[0088] GraphPad Prism 9 software was used to analyze the differences in growth indicators, chromaticity values, astaxanthin content, immune and antioxidant and related gene expression level parameters of each group; data were expressed as "mean ± standard error", P < 0.05 indicated a significant difference, and P < 0.01 indicated an extremely significant difference.

[0089] like Figure 2 As shown, a detection system for the effect of natural astaxanthin on the growth, body color and immunity of Macrobrachium rosenbergii provided by an embodiment of the present invention comprises:

[0090] A collection module, used for collecting samples;

[0091] A calculation module, used for calculating growth indicators;

[0092] The measurement module is used for body color measurement, immune and antioxidant enzyme activity measurement, immune and molting gene expression level measurement, and astaxanthin content measurement.

[0093] The analysis module is used to perform differential analysis on growth indexes, chromaticity values, astaxanthin content, immunity and antioxidant and related gene expression level parameters of each group using GraphPadPrism 9 software.

[0094] Another object of the present invention is to provide a computer device, comprising a memory and a processor, wherein the memory stores a computer program, and when the computer program is executed by the processor, the processor executes the steps of the method for testing the effect of natural astaxanthin on the growth, body color and immunity of Macrobrachium rosenbergii.

[0095] Another object of the present invention is to provide a computer-readable storage medium storing a computer program, which, when executed by a processor, enables the processor to execute the steps of the method for testing the effect of natural astaxanthin on the growth, body color and immunity of Macrobrachium rosenbergii.

[0096] Another object of the present invention is to provide an information data processing terminal, which is used to implement a detection system for the effect of natural astaxanthin on the growth, body color and immunity of Macrobrachium rosenbergii.

[0097] The present invention is specifically implemented:

[0098] 1. The present invention feeds different amounts of natural astaxanthin to detect and evaluate its effects on the growth, immunity, antioxidant enzyme activity and related gene expression of Macrobrachium rosenbergii, in order to provide a reference for the use of astaxanthin in the healthy cultivation of Macrobrachium rosenbergii.

[0099] 2. Materials and Methods

[0100] 2.1. Experimental materials and design

[0101] This experiment was conducted in a circulating aquarium (130 cm × 58 cm × 40 cm) at the Pearl River Fisheries Research Institute of the Chinese Academy of Fishery Sciences (E: 113°22′, N: 23°06′). The initial body weight of the Macrobrachium rosenbergii used in the experiment was 0.32±0.02 g. 800 shrimps were randomly divided into 4 groups, and the amount of natural astaxanthin added to each group was 0 (Group C), 50 mg kg -1 (Ax50 group), 100 mg kg -1 (Ax100 group) and 200 mg kg -1 (Ax200 group), with 4 replicates per group. A commercial feed without astaxanthin (Huolibao, Zhuhai, Guangdong, China) was used as the basic feed group, i.e., the control group (Group C).

[0102] The feeding amount was 7-9% of body weight from 1st to 10th day (Sun et al., 2020; Fang et al., 2022;), and 5% of body weight from 11th to 20th day (Zhuang et al., 2022). Feeding was done twice a day, 40% and 60% of the daily feeding amount in the morning (9:00) and afternoon (18:00), respectively. Sewage was sucked out and water (1 / 4) was changed before feeding every morning. Water quality (ammonia nitrogen, nitrite, residual chlorine, pH, total alkali and total hardness) was tested three times a week, and the water temperature was controlled at 28-32°C. The molting cycle of Macrobrachium rosenbergii was recorded during the culture process.

[0103] Sample collection and analysis

[0104] 2.2.1. Sample collection

[0105] The sample collection operation was carried out in accordance with the animal collection rules of the Animal Experiment Ethics Committee of the Pearl River Fisheries Research Institute, fully respecting animal welfare. Fasting for 24 hours before each sampling. Sampling was performed in the 10th and 20th weeks of culture. The number of surviving Macrobrachium rosenbergii in each group was counted and the survival rate was calculated; 20 tails were randomly picked from each group to count the weight and body length; 12 live shrimps were randomly taken from each group for live shrimp body color determination, and in the 20th week, 8 shrimps were taken from each group for 3 minutes in boiling water bath for determination of cooked shrimp body color. 12 Macrobrachium rosenbergii were taken from each group (every 3 shrimp tissues were mixed into 1 portion, a total of 4 portions, the same below) and hemolymph was extracted along the base of the fifth step foot with a 1mL disposable syringe containing anticoagulant, centrifuged at 1000xg for 5 minutes, and the supernatant was aspirated and stored at -80℃. The hepatopancreas was taken and weighed for calculation of the liver-to-body ratio and evaluation of subsequent related enzyme activities and expression levels of immune-related genes. Muscles from the 20th week were also taken for detection of astaxanthin content.

[0106] According to the structural changes in the distal region of the fifth abdominal foot, the molting process of each group of Macrobrachium rosenbergii was divided into three periods: intermolting, premolting and postmolting (Peebles, 1977; Kamaruding et al., 2017). Four hepatopancreas samples (3 tails / sample) were taken from each molting period to evaluate the expression levels of related genes in different treatment groups at different molting periods.

[0107] All hepatopancreas and muscle tissues were frozen with liquid nitrogen and then stored at -80°C for subsequent determination of various indicators.

[0108] 2.2.2. Growth index calculation

[0109] At the end of the 10th and 20th weeks, the body length, body weight and hepatopancreas weight of Macrobrachium rosenbergii were measured using tpsDig2 software (F. James Rohlf, Stony Brook University, Stony Brook, NY, USA) and a Mettler Toledo AL-204 precision balance (Mettler Toledo, Inc., Shanghai, China). The following formulas were used to calculate growth index parameters such as survival rate, weight gain rate, specific growth rate and liver-to-body ratio:

[0110] Survival rate (SR,%) = N t / N0×100

[0111] Weight gain rate(WGR,%)=(W t -W0) / W0×100

[0112] Specific growth rate (SGR,% / d)=(lnW t -lnW0) / t

[0113] Hepatosomatic index(HSI,%)=W h / W b ×100

[0114] Where: N0 is the initial number (tail); N t is the final weight (tail); W0 is the initial weight (g); W t is the final body weight (g); t is the number of culture days (d); W h W is the weight of hepatopancreas (g); b is body weight (g).

[0115] 2.2.3. Body color determination

[0116] At the 10th and 20th weeks of culture, the L of Macrobrachium rosenbergii was measured using a 3nh colorimeter (Shenzhen Sanlian Technology Co., Ltd., Shenzhen, China). * value (represents brightness, the larger the value, the lighter the color), a * Value(+a * Reddish, -a * Greenish) and b * Value(+b * Yellowish, -b * The color difference ΔE was calculated by the following formula:

[0117]

[0118] Where: L * is the brightness of the treatment group; is the brightness of the control group; a * is the redness of the treatment group, b is the redness of the control group; * is the yellowness of the treatment group, Yellowness of the control group.

[0119] 2.2.4. Immunity and antioxidant enzyme activity assays

[0120] The activities of superoxide dismutase (SOD), catalase (CAT), malondialdehyde (MDA), alkaline phosphatase (AKP) and lysozyme (LZM) in the hepatopancreas and hemolymph of Macrobrachium rosenbergii at the end of the 10th and 20th weeks were determined by WST-1 method, ammonium molybdate method, thiobarbital acid (TBA) reaction method, microenzyme labeling method and turbidimetry, respectively, to evaluate the effects of astaxanthin on immunity and antioxidant. The operation steps were in accordance with the instructions of the kit of Nanjing Jiancheng Bioengineering Institute (Nanjing, China).

[0121] 2.2.5. Determination of immune and molting gene expression levels

[0122] Total RNA from hepatopancreas was extracted by Trizol (Invitrogen, Waltham, MA, USA), and the integrity of RNA was evaluated by 1% agarose gel electrophoresis. The concentration of RNA was detected by Nanodrop-2000 spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA, USA), and cDNA was synthesized according to the instructions of M-MLV reverse transcription kit (Invitrogen, Waltham, MA, USA). Real-time fluorescence quantitative PCR was performed using StepOnePlus Real-Tim PCR system (Applied Biosystems, Foster City, CA, USA) to evaluate the expression levels of immune and molting-related genes in Macrobrachium rosenbergii after astaxanthin addition. The reaction system included 1 μL (50 ng / μL) cDNA, 10 μL 2×SYBR Green Premix (Universal), 1 μL primer (10 pmol / μL) and 7 μL double distilled water, with a total system of 20 μL. The reaction procedure was as follows: pre-denaturation at 95°C for 30 min; 35 cycles of denaturation at 95°C for 40 s, annealing at 60°C for 45 s, and extension at 72°C for 10 min; data acquisition at 72°C for 10 min, and then melting curves were obtained at 95°C for 5 s, 60°C for 30 s, and 95°C for 15 s.

[0123] 2.2.6. Determination of astaxanthin content

[0124] 5 g muscle sample was extracted with acetonitrile, defatted and concentrated with n-hexane, and the astaxanthin content in the muscle was detected by HPLC (Shanghai Zhuangrun International Trade Co., Ltd., Shanghai, China) (refer to (SN / T2327-2009) method).

[0125] Statistical analysis

[0126] GraphPad Prism 9 software (GraphPad Software, San Diego, CA) was used to analyze the growth index, color value, astaxanthin content, immune and antioxidant and related gene expression level parameters of each group. Data are expressed as "mean ± standard error", P < 0.05 indicates a significant difference, and P < 0.01 indicates an extremely significant difference.

[0127] 3. Results

[0128] 3.1. Effects of natural astaxanthin on the growth performance of Macrobrachium rosenbergii

[0129] Effects of different astaxanthin additions on growth indicators of Macrobrachium rosenbergii. The results of growth indicators at week 10 showed that the body weight of the Ax200 group was significantly higher than that of the Ax100 group (p<0.05) and extremely significantly higher than that of the control group (p<0.01), while the Ax50 group was significantly higher than that of the control group (p<0.05), but there was no significant difference with the Ax100 group. The body length of the Ax200 group was significantly higher than that of the control group (p<0.05). In terms of weight gain rate, both the Ax50 and Ax100 groups were significantly higher than the control group (p<0.05), but there was no significant difference with the Ax200 group; the specific growth rates of the three astaxanthin groups were significantly higher than those of the control group (p<0.05). The liver-to-body ratio of the Ax200 group was significantly lower than that of the control group (p<0.05); there was no significant difference in survival rate among the groups.

[0130] The growth results of Macrobrachium rosenbergii in the 20th week showed that the body weights of the three groups of Macrobrachium rosenbergii fed with astaxanthin were significantly higher than those of the group not fed with astaxanthin (p<0.05); in terms of body length, the body length of the control group was significantly lower than that of the Ax100 group (p<0.05) and extremely significantly lower than that of the Ax200 group (p<0.01). In terms of weight gain rate, the weight gain rates of the Ax50 and Ax100 groups were significantly higher than those of the control group (p<0.05), but there was no significant difference with the Ax200 group. The specific growth rates of the Ax100 and Ax200 groups were significantly higher than those of the control group and the Ax50 group (p<0.05). In terms of survival rate, the survival rate of the Ax50 group was significantly higher than that of the other three groups (p<0.05). There was no significant difference in the liver-to-body ratio among the groups. The molting period of the Ax100 group was significantly shorter than that of the control group (p<0.05).

[0131] 3.2. Effect of natural astaxanthin on the body color of Macrobrachium rosenbergii

[0132] The results of the body color of live Macrobrachium rosenbergii at 10 weeks under different astaxanthin additions are shown in the attached Figure 1 The L * The values ​​were significantly higher than those of Ax100 and Ax200 groups (p<0.05) and extremely significantly higher than those of Ax50 group (p<0.01). * Value, b * The value was significantly higher than that of the control group (p<0.05) and extremely significantly higher than that of the Ax50 group (p<0.01); however, the ΔE value showed that there was no significant difference in the color difference of Macrobrachium rosenbergii fed with astaxanthin. Figure 1 The L of each treatment group * The values ​​of Ax200 group were significantly lower than those of the control group (p<0.05). * The values ​​were significantly higher than those of the other three groups (p<0.05). *The ΔE values ​​of the two high-dose groups fed with astaxanthin (ΔE Ax100-C and ΔE Ax200-C ) was significantly higher than the ΔE value of the low-dose group (ΔE Ax50-C )(p<0.05), while there was no significant difference between the two high-dose groups. Figure 2 The L * The value of Ax200 group was significantly higher than that of Ax100 group (p<0.05) and extremely significantly higher than that of Ax200 group (p<0.01). * The value of the control group was significantly higher than that of the other three groups (p<0.05). * The value was significantly lower than that of Ax50 and Ax100 groups (p<0.05), and extremely significantly lower than that of Ax200 group (p<0.01). Ax200-C Significantly higher than ΔE Ax50-C (p<0.05), and significantly higher than ΔE Ax100-C (p<0.01), and ΔE Ax50-C and ΔE Ax100-C There was a significant difference (p<0.05). With the increase of astaxanthin dosage in the basic feed, the color of 20-week-old Macrobrachium rosenbergii (cooked) gradually deepened.

[0133] Astaxanthin content in muscle of Macrobrachium rosenbergii at week 20 with different astaxanthin additions. After adding astaxanthin, the astaxanthin content in muscle increased significantly compared with the control group (p<0.05).

[0134] 3.3. Effects of natural astaxanthin on the immune and antioxidant capacity of Macrobrachium rosenbergii

[0135] Effects of different astaxanthin additions on hemolymph immunity and antioxidant enzyme activities of Macrobrachium rosenbergii. The results of enzyme activity at the 10th week of culture showed that the SOD activity of the Ax100 group was significantly lower than that of the control group and the Ax200 group (p<0.05); the LZM activity of the Ax50 group was significantly higher than that of the Ax200 group (p<0.05); the AKP activity of the Ax200 group was significantly higher than that of the control group (p<0.05); while there were no significant differences in MDA and CAT activities among the groups. The results of enzyme activity at the 20th week of culture showed that the SOD activity of the Ax200 group was significantly higher than that of the control group and the Ax50 group (p<0.05), and there was no significant difference in SOD activity between the Ax100 group and the other three groups; the MDA and AKP activities of the Ax200 group were significantly higher than those of the other three groups (p<0.05); while there were no significant differences in CAT and LZM activities among the groups.

[0136] Effects of different astaxanthin additions on immune and antioxidant enzyme activities in the hepatopancreas of Macrobrachium rosenbergii. The results of enzyme activity at week 10 showed that the SOD activity of the Ax100 group was significantly lower than that of the other three groups (p<0.05); the MDA and AKP activities of the group not fed with astaxanthin were significantly higher than those of the other three groups (p<0.05); the CAT activity of the Ax50 group was significantly higher than that of the other three groups (p<0.05); there was no significant difference in LZM activity among the groups. The results of enzyme activity at week 20 showed that the MDA activity of the group not fed with astaxanthin was significantly higher than that of the Ax100 group (p<0.05); there was no significant difference in the activities of SOD, CAT, LZM and AKP among the groups.

[0137] 3.4. Effects of natural astaxanthin on the expression levels of immune and antioxidant genes in Macrobrachium rosenbergii

[0138] Results of hepatopancreatic immune and antioxidant gene expression levels in Macrobrachium rosenbergii at 10 weeks under different astaxanthin supplementation levels. The cat gene expression level in the Ax100 group was significantly lower than that in the other three groups (p<0.05). The sod gene expression level in the Ax50 group was significantly higher than that in the other three groups (p<0.05). The gsh-px gene expression level in the Ax200 group was significantly lower than that in the other three groups (p<0.05). The acp gene expression level in the Ax100 group was significantly higher than that in the Ax50 and Ax200 groups (p<0.05), but there was no significant difference compared with the group not fed astaxanthin.

[0139] Results of hepatopancreatic immune and antioxidant gene expression levels at week 20. The cat gene expression level in the Ax100 group was significantly higher than that in the control group (p<0.05). The sod gene expression levels in the Ax100 and Ax200 groups were significantly higher than those in the control group and Ax50 group (p<0.05). The gsh-px gene expression level in the control group was significantly lower than that in the Ax100 group (p<0.05) and extremely significantly lower than that in the Ax50 group (p<0.01), while that in the Ax50 group was significantly higher than that in the Ax200 group (p<0.05). The acp gene expression level in the Ax100 group was significantly higher than that in the control group and Ax50 group (p<0.05).

[0140] 3.5. Effects of natural astaxanthin on the expression level of molting genes in Macrobrachium rosenbergii

[0141] The results of molting gene expression in Macrobrachium rosenbergii at different molting stages after being fed astaxanthin for 10 weeks. In the late molting period, the gene expression level of MIH gene in Ax50 group was significantly higher than that in the other three groups (p<0.05); in the intermolting period, the gene expression level of Ax50 group was significantly higher than that in Ax100 and Ax200 groups (p<0.05), and extremely significantly higher than that in the control group (p<0.01); in the early molting period, the gene expression level of Ax50 group was significantly higher than that in Ax200 group (p<0.05), and extremely significantly higher than that in Ax50 group (p<0.01). The gene expression level of EcR gene in the Ax50 group was significantly higher than that of the other three groups in the late molting period (p<0.05); in the intermolting period, the Ax200 group was significantly higher than the two low-dose groups (p<0.05) and extremely significantly higher than the unfed group (p<0.01); in the early molting period, the Ax100 and Ax200 groups were significantly higher than the low-dose and unfed groups (p<0.05). The gene expression level of RXR gene in the Ax50 group was significantly higher than that of the other three groups in the late molting period (p<0.05); in the intermolting period, the high-dose group was significantly higher than the two low-dose groups (p<0.05) and extremely significantly higher than the control group (p<0.01); in the early molting period, the high-dose group was significantly higher than the control group and the Ax50 group (p<0.05).

[0142] Gene detection results after 20 weeks: In the late molt period, the gene expression level of MIH gene in Ax200 group was significantly lower than that in the other three groups (p<0.05); in the intermolt period, the gene expression level of Ax50 and Ax200 groups was significantly lower than that in Ax100 group (p<0.05); while in the early molt period, the gene expression level of control group was significantly lower than that of Ax50 and Ax100 groups (p<0.05). In the late molt period, the gene expression level of EcR gene in Ax100 group was significantly higher than that in Ax50 group (p<0.05) and extremely significantly higher than that in the other two groups (p<0.01); in the intermolt period, the gene expression level of Ax100 group was significantly higher than that in control group and Ax200 group (p<0.05) and extremely significantly higher than that in Ax50 group (p<0.01); in the early molt period, the gene expression level of Ax50 group was significantly higher than that in control group and Ax200 group (p<0.05), and there was no significant difference among the other three groups. In the late molt period, the gene expression level of RXR gene in Ax50 group was significantly lower than that in Ax200 group (p<0.05); in the intermolt period, the gene expression level in the control group was significantly higher than that in the other three groups (p<0.05); in the early molt period, there was no significant difference in gene expression level among the groups.

[0143] 4. Discussion

[0144] 4.1. Effects of natural astaxanthin on the growth performance of Macrobrachium rosenbergii

[0145] There have been many reports on studies on astaxanthin significantly improving the growth performance of crustaceans. For example, when 0.6% (astaxanthin content: 126.60 mg kg-1) and 1.2% (astaxanthin content: 253.20 mg kg-1) of Haematococcus pluvialis were added to the feed and fed to Procambarus clarkii, their final body weight, weight gain rate and specific growth rate were significantly higher than those of the basic feed group (Cai et al., 2022); when fed a feed containing 160 mg kg-1 of astaxanthin, the weight gain rate and specific growth rate of Penaeus vannamei increased by 71.86% and 0.17%, respectively (Zhao et al., 2022). The results of this study showed that the addition of astaxanthin can improve the weight gain rate and specific growth rate of Macrobrachium rosenbergii juveniles at both the 10th and 20th weeks, especially when the astaxanthin feeding amount of 100 mg kg-1 was fed, its growth performance was the best overall. Xu et al. (2022) added 100 and 200 mg kg-1 astaxanthin to the feed and fed Macrobrachium rosenbergii for 10 weeks. The results showed that the weight gain rate and specific growth rate of Macrobrachium rosenbergii fed with astaxanthin were slightly higher than those of the unfed group, but not significantly. Their results were inconsistent with this study, but it was proved that the addition of astaxanthin can increase the weight gain rate and specific growth rate in Penaeus monodon (Niu et al., 2012) and Penaeus japonicus (Wang et al., 2018). This paper speculates that the difference in results may be due to differences in basic feed ingredients and feeding patterns (Long et al., 2023). In terms of molting cycle, the results of this study showed that the molting cycle of Macrobrachium rosenbergii was significantly shortened by adding 100 mg kg-1 astaxanthin. Moreover, the research results of Flores et al. (2007) showed that adding 80 mg kg-1 astaxanthin can increase the molting frequency of Penaeus vannamei and shorten the molting cycle, indicating that adding astaxanthin can significantly promote the molting of Macrobrachium rosenbergii.

[0146] 4.2 Effects of natural astaxanthin on body color and astaxanthin content of Macrobrachium rosenbergii

[0147] The body color of aquatic animals varies with the pigment and content of the food they ingest (Lorenz and Cysewski, 2000; Lim et al., 2018). Studies have shown that astaxanthin can promote pigmentation and change the body color of aquatic organisms. Adding 0.2% of Haematococcus lacustris (3% astaxanthin content) can increase the brightness of Plectropomus leopardus (L * ) was significantly reduced, and the redness (a * ) and yellowness (b *) was significantly increased (Zhang et al., 2023). -1 Astaxanthin feeding of blood parrotfish also significantly reduced brightness and significantly increased redness and yellowness (Micah et al., 2022). In addition, supplementation with natural astaxanthin significantly improved the brightness, redness and yellowness of cooked giant tiger shrimp (Penaeus monodon), and its effect was better than synthetic astaxanthin (Angell et al., 2018). Similarly, in the present invention, compared with the control group, natural astaxanthin reduced the brightness L of Macrobrachium rosenbergii at the 10th and 20th weeks. * ; The redness and yellowness values ​​of the Ax100 and Ax200 groups at the 10th week were significantly higher than those of the control group; the redness and yellowness values ​​of the Ax200 group at the 20th week were significantly higher than those of the control group; at the 20th week, the color difference value (ΔE) between the live shrimps of each treatment group and the control group increased with the increase of astaxanthin addition, indicating that different astaxanthin additions can change the body color of Macrobrachium rosenbergii. This may be because natural astaxanthin mainly exists in the form of esterification, which is beneficial to the deposition of pigments in the body after ingestion. Crustaceans lack the ability to synthesize astaxanthin from scratch and can only ingest natural astaxanthin from food or ingest carotenoids to synthesize astaxanthin to meet the needs of the body. Astaxanthin deficiency can cause the body color of Procambarus clarkii to turn blue and the color of red pigment cells to fade, but supplementation with astaxanthin can restore its normal body color (Guo, 2023). In addition, the astaxanthin content in muscle is considered to be an important indicator for evaluating the utilization rate of astaxanthin in organisms (Kiessling et al., 2006). The present invention shows that the astaxanthin content in the muscle of Macrobrachium rosenbergii increases with the increase of the amount of natural astaxanthin added, which is similar to the results of red sea bream (Pagruspagrus) (Tejera et al., 2007), giant tiger shrimp (Huang et al., 2023), and vannamei shrimp (Zhao et al., 2022), indicating that the enrichment of astaxanthin in crustaceans is homogeneous. In summary, the present invention believes that the astaxanthin addition amount in the feed is 100 and 200 mg kg -1 When the astaxanthin in the shell and muscle of Macrobrachium rosenbergii accumulates more, the body color changes more significantly.

[0148] 4.3 Effects of natural astaxanthin on immunity and antioxidant enzyme activities of Macrobrachium rosenbergii

[0149] Crustaceans lack the specific immune system possessed by vertebrates and can only resist the invasion of pathogens through the nonspecific immune system (Huang and Ren, 2020; Kulkarni et al., 2021). At present, measuring the activity levels of immune and antioxidant enzymes in hemolymph and hepatopancreas is one of the conventional methods to evaluate changes in the immune capacity of experimental animals. Acid phosphatase (AKP) and lysozyme (LZM) are important parameters for evaluating the nonspecific immunity of crustaceans. Studies have shown that astaxanthin can enhance immune performance by increasing the AKP activity of hemolymph of Procambarus clarkii (Cheng and Wu, 2019) and red claw crayfish (Cherax quadricarinatus) juveniles (Cheng et al., 2024). The results of hemolymph AKP activity in the treatment group of the present invention are consistent with the above results. AKP activity increased with the increase of astaxanthin addition in both the 10th and 20th weeks, and the Ax200 group was significantly higher than the control group. The results of AKP activity in the hepatopancreas of the present invention showed that the AKP activity in the 10th week treatment group was significantly lower than that in the control group, which is the same as the results of Xu Junjie et al. (Xu et al., 2022). The hepatopancreas is the main metabolic organ of shrimp (Duan et al., 2023), and many generations of physiological metabolism are carried out in this organ. As a non-specific phosphohydrolase, AKP regulates the metabolic activities of aquatic animals by catalyzing the hydrolysis of monophosphates and the transfer of phosphate groups (Zhang and Zhang, 2003). The decrease in the AKP enzyme activity in the hepatopancreas of the present invention may be because most of it is used for physiological metabolic activities. At the same time, AKP can increase the level of stress resistance and stress resistance by enhancing its activity, and can also promote the absorption of calcium and the secretion of chitin by shrimp, thereby promoting molting growth (Cheng and Wu, 2019). The decrease in the hepatopancreas enzyme activity in the 10th week treatment group of the present invention may be related to astaxanthin promoting crustacean molting and accelerating calcification. As one of the important indicators of the nonspecific immune system, LZM can activate phagocytes in the immune defense of crustaceans and effectively kill Gram-positive bacteria ( and Peters, 1990). For example, the addition of astaxanthin to feed enhanced the LZM activity of Procambarus clarkii (Cheng and Wu, 2019). The results of hemolymph in the 10th week of the present invention showed that the LZM activity first increased and then decreased with the increase of astaxanthin addition, and the Ax200 group was significantly lower than the Ax50 group. Astaxanthin had no significant effect on the LZM activity of Penaeus vannamei (Wang et al., 2019), which is consistent with the LZM activity results of hemolymph in the 20th week and hepatopancreas in the 10th and 20th weeks of the present invention. It shows that astaxanthin is beneficial to improve the immune capacity of the hemolymph tissue of Macrobrachium rosenbergii.

[0150] As a strong antioxidant, astaxanthin can regulate the redox balance of biological cells, control the fluctuation of antioxidant enzyme activity and oxidation product levels, and effectively remove reactive oxygen species (ROS) in the body, preventing further oxidation reactions and pathological changes (Rahman et al., 2006; Cheng and Wu, 2019). SOD, MDA and CAT are important indicators for evaluating the antioxidant capacity of organisms (Wang et al., 2012; Leite et al., 2021; Dawood et al., 2022). The synergistic effect of SOD and CAT can convert reactive oxygen in the body into H2O2 and then decompose it into H2O. In addition, astaxanthin contains a violet ring that can remove reactive oxygen in crustaceans (Ambati et al., 2014; Fang et al., 2022). In the present invention, the SOD activity in the hemolymph and hepatopancreas at the 10th week first decreased and then increased, and the Ax100 group was significantly lower than the control group and the Ax200 group; at the 20th week, the SOD activity in the hemolymph of the Ax200 group was significantly higher than the control group and the Ax50 group. As for the CAT activity, only the Ax50 group in the hepatopancreas at the 10th week was significantly higher than the other three groups. This may be because SOD and CAT are consumed to remove excess reactive oxygen in the body, and the SOD and CAT enzyme activities increase with the addition of astaxanthin. MDA is a reaction product of lipid peroxidation in organisms, and an increase in its value indicates that the body is more damaged ( et al., 2000; Mathew et al., 2007; Cheng and Wu, 2019). In this experiment, the MDA activity of the Ax200 group in the hemolymph at week 20 was significantly higher than that of the other three groups; in the hepatopancreas, the MDA activity of the treated group at week 10 was significantly reduced compared with the control group, and the MDA activity of the Ax100 group at week 20 was significantly reduced, which indicated that the addition of astaxanthin at 50 and 100 mg kg -1 It can more effectively remove free radicals in the body of Macrobrachium rosenbergii and maintain redox balance.

[0151] While astaxanthin changes the hepatopancreas's immune and antioxidant enzyme activities to resist pathogen invasion and remove reactive oxygen species, its antioxidant gene expression levels will also change accordingly. Studies have shown that astaxanthin reduces the expression of sod, gsh-px and cat in the hepatopancreas of white shrimp (Fang et al., 2022). The reason for this parameter change is that astaxanthin itself can remove reactive oxygen species in crustaceans, so there is no need to produce more antioxidant enzymes (Ambati et al., 2014; Xue et al., 2023). The results of the 10th week of the present invention showed that the cat gene decreased with the increase in the amount of astaxanthin added, with the lowest expression in the Ax100 group and then rising; the sod gene expression level in the Ax50 group was significantly higher than that in the other three groups, indicating that the addition of 50 mg kg -1 The expression of sod gene was enhanced, and the antioxidant capacity and immune capacity were improved. The addition amount was 100 and 200 mg kg -1 When treated with high doses of astaxanthin, it may have directly activated the nonspecific immune response of crustaceans (Ambati et al., 2014; Fang et al., 2022); the expression level of gsh-px gene in the Ax200 group was significantly lower than that in the other three groups; the expression level of acp gene in the Ax100 group was the highest, significantly higher than that in the Ax50 and Ax200 groups.

[0152] The results of the present invention show that after adding astaxanthin, the expression level of cat in the 20th week showed a trend of first increasing and then decreasing, and the Ax100 group was significantly higher than the control group; the sod gene expression levels of the Ax100 group and the Ax200 group were significantly higher than the control group and the Ax50 group; the gsh-px gene expression level also showed a trend of first increasing and then decreasing, and the expression level of the Ax50 group was the highest and significantly higher than the control group; the acp gene expression level showed the same trend as the cat gene expression level, indicating that the addition of astaxanthin may significantly improve the immune and antioxidant capacity of Macrobrachium rosenbergii by changing the expression levels of the immune and antioxidant genes of Macrobrachium rosenbergii, and the astaxanthin addition amount was 100 mg / kg -1 The effect is best when

[0153] The molting of crustaceans is divided into growth molting and reproductive molting, and is regulated by molting-related hormones and their encoding genes. During the molting process, molt-inhibiting hormone (MIH) plays a major role by antagonizing molting hormone (Lachaise et al., 1993; De Kleijn and Van Herp, 1995). MIH can regulate the duration of the molting cycle by inhibiting the synthesis of molting steroids (Watson and Spaziani, 1985; Okumura et al., 2005; Huang et al., 2015). When astaxanthin was added at a low dose, the MIH expression level of Macrobrachium rosenbergii was significantly higher than that of the control group, and was significantly lower than that of the control group when astaxanthin was added at a high dose (Xu et al., 2024). The results of the present invention show that in the 10th week, the expression of the MIH gene in the Ax50 group of Macrobrachium rosenbergii was significantly higher than that in the control group at each molting period, indicating that low-dose astaxanthin may inhibit the steroid production of Macrobrachium rosenbergii compared with the control group, thereby delaying or inhibiting molting. The EcR gene is a candidate gene for regulating growth (Qiu et al., 2020), and is also involved in and mediates the ovarian development and yolk protein synthesis process (Hiraoka and Shastry, 2000). As a member of the nuclear receptor family, the RXR gene plays an important role in regulating individual physiological activities such as cell differentiation, growth and development, and metabolism (Chen et al., 2018; et al., 2019; Li et al., 2021). Macrobrachium rosenbergii regulates ecdysone levels through the EcR / RXR polymer complex formed by EcR and RXR (Clayton et al., 2001; Nakagawa and Henrich, 2009). The research results of Xu et al. (Xu et al., 2024) showed that the expression level of EcR in Macrobrachium rosenbergii first decreased and then increased with the increase of astaxanthin addition, which is similar to the EcR result of the present invention at week 20. The expression levels of the EcR gene and RXR gene of the present invention are both affected by astaxanthin, and the change trends of the two genes are basically the same. The two may combine to form a polymer to regulate ecdysone and thus mediate the molting of Macrobrachium rosenbergii (Thummel, 1995).

[0154] Astaxanthin was added at 100 and 200 mg kg -1 It can significantly improve the growth performance and body color of Macrobrachium rosenbergii and increase the astaxanthin content in muscle. At the same time, astaxanthin can improve the immunity and antioxidant capacity of Macrobrachium rosenbergii. However, the factors affecting the absorption of astaxanthin by Macrobrachium rosenbergii need further study. The results of this study provide reference data for the application of astaxanthin in crustacean feed and the promotion of the healthy development of aquaculture.

[0155] Example 1: Optimization of shrimp nutrition formula in aquaculture

[0156] In modern aquaculture, farmers often need to optimize feed formulas in order to increase the growth rate, enhance body color and improve immunity of Macrobrachium rosenbergii. The present invention provides accurate data support for aquatic feed companies by systematically testing and evaluating the effects of natural astaxanthin on the growth, body color and immune system of Macrobrachium rosenbergii. In practical applications, feed companies can develop functional feeds rich in astaxanthin based on the research results of the present invention, and improve the market value and survival rate of shrimp by enhancing the body color and immunity of shrimp. Through this optimized formula, feed companies can provide more competitive products to farms and improve the economic benefits of aquaculture.

[0157] Example 2: Product quality control in aquatic product processing enterprises

[0158] Aquatic product processing companies have high requirements for the appearance quality and antioxidant properties of shrimps, especially when shrimp products are exported, body color and antioxidant properties are important criteria for evaluating the quality of shrimps. The astaxanthin content and body color change evaluation method provided by the present invention can help aquatic product processing companies to monitor the growth and body color performance of shrimps in real time during the breeding and processing process. By reasonably adding astaxanthin during the breeding process, it is ensured that the processed shrimp products have an ideal appearance color and have strong antioxidant properties, thereby extending the shelf life of the products and improving product quality. During the refrigeration, transportation and sales of shrimps, companies can use the colorimeter and other detection technologies in the present invention to ensure that the products meet the export standards and further enhance the international market competitiveness of the products.

[0159] These two examples show that the present invention has a wide range of application potentials in the field of aquaculture and processing, and can effectively improve the overall economic benefits of the industrial chain. It should be noted that the embodiments of the present invention can be implemented by hardware, software, or a combination of software and hardware. The hardware part can be implemented using dedicated logic; the software part can be stored in a memory and executed by an appropriate instruction execution system, such as a microprocessor or dedicated design hardware. A person of ordinary skill in the art will understand that the above-mentioned devices and methods can be implemented using computer executable instructions and / or included in a processor control code, such as a carrier medium such as a disk, CD or DVD-ROM, a programmable memory such as a read-only memory (firmware), or a data carrier such as an optical or electronic signal carrier. Such code is provided on the carrier medium. The device and its modules of the present invention can be implemented by hardware circuits such as ultra-large-scale integrated circuits or gate arrays, semiconductors such as logic chips, transistors, etc., or programmable hardware devices such as field programmable gate arrays, programmable logic devices, etc., can also be implemented by software executed by various types of processors, and can also be implemented by a combination of the above-mentioned hardware circuits and software, such as firmware.

[0160] The above description is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Any modifications, equivalent substitutions and improvements made by any technician familiar with the technical field within the technical scope disclosed by the present invention and within the spirit and principle of the present invention should be covered by the protection scope of the present invention.

Claims

1. A method for testing the effect of natural astaxanthin on the growth, body color and immunity of Macrobrachium rosenbergii, characterized in that: During the shrimp sample collection process, we strictly followed the experimental ethical requirements and accurately controlled the sampling time and methods; used efficient formulas to calculate growth indicators such as survival rate, weight gain rate, specific growth rate and liver-to-body ratio; used a precise colorimeter to measure the body color parameters of Macrobrachium rosenbergii, and calculated the color difference value using the color difference formula; used standardized kits to determine the activity of immune and antioxidant enzymes, and combined with real-time fluorescence quantitative PCR technology to evaluate the expression levels of immune and molting genes; used high-performance liquid chromatography to detect the astaxanthin content in the muscle, and finally used professional statistical software for differential analysis to ensure the accuracy of the data and the reliability of the research results.

2. The method for testing the effect of natural astaxanthin on the growth, body color and immunity of Macrobrachium rosenbergii as claimed in claim 1, characterized in that: The following steps are involved: Step 1, sample collection; Step 2, growth index calculation; Step 3, body color determination; immune and antioxidant enzyme activity determination; immune and molting gene expression level determination; astaxanthin content determination; Step 4: Statistical analysis of data.

3. The method for testing the effect of natural astaxanthin on the growth, body color and immunity of Macrobrachium rosenbergii as claimed in claim 2, characterized in that: Collection of the samples: Sample collection was carried out in accordance with the animal collection rules of the Animal Experiment Ethics Committee of the Pearl River Fisheries Research Institute; fasting for 24 hours before each sampling; sampling was performed in the 10th and 20th weeks of breeding; the number of surviving Macrobrachium rosenbergii in each group was counted and the survival rate was calculated; 20 shrimps were randomly collected from each group to count the weight and body length; 12 live shrimps were randomly taken from each group for live shrimp body color determination, and in the 20th week, 8 shrimps were taken from each group and boiled in water for 3 minutes to determine the body color of cooked shrimps; 12 Macrobrachium rosenbergii were taken from each group; tissues of every 3 shrimps were mixed into 1 portion, for a total of 4 portions, the same below; hemolymph was extracted along the base of the fifth stepping foot with a 1mL disposable syringe containing an anticoagulant, centrifuged at 1000xg for 5 minutes, and the supernatant was aspirated and stored at -80℃; the hepatopancreas was taken and weighed for calculation of the liver-to-body ratio and evaluation of subsequent related enzyme activities and expression levels of immune-related genes; muscles from the 20th week were taken for detection of astaxanthin content; According to the structural changes in the distal region of the fifth abdominal foot, the molting process of Macrobrachium rosenbergii in each group was divided into three periods: intermolting, premolting and postmolting. Four hepatopancreas samples were collected during each molting period; 3 samples / sample were used to evaluate the expression levels of related genes in different treatment groups at different molting periods. All hepatopancreas and muscle tissues were frozen with liquid nitrogen and then stored at -80°C for subsequent determination of various indicators.

4. The method for testing the effect of natural astaxanthin on the growth, body color and immunity of Macrobrachium rosenbergii as claimed in claim 2, characterized in that: The growth index calculation: After the breeding experiment, the following formulas were used to calculate growth index parameters such as survival rate, weight gain rate, specific growth rate and liver-to-body ratio: Survival rate(SR,%)=N t / N0×100 Weight gain rate(WGR,%)=(W t -W0) / W0×100 Specific growth rate(SGR,% / d)=(lnW t -lnW0) / p Hepatosomatic index(HSI,%)=W h / W b ×100 Where: N0 is the initial number (tail); N t is the final quantity (tail); W0 is the initial body weight (g); W t is the final body weight (g); t is the number of culture days (d); W h W is the weight of hepatopancreas (g); b is body weight (g).

5. The method for testing the effect of natural astaxanthin on the growth, body color and immunity of Macrobrachium rosenbergii as claimed in claim 1, characterized in that: The body color determination: At the 10th and 20th weeks of culture, the L of Macrobrachium rosenbergii was measured using a 3nh colorimeter (Shenzhen Sanlian Technology Co., Ltd., Shenzhen, China). * value (represents brightness, the larger the value, the lighter the color), a * Value(+a * Reddish, -a * Greenish) and b * Value(+b * Yellowish, -b * The color of the cooked shrimp was measured at the 1st, 3rd and 5th abdominal segments (Zhang et al., 2021). In addition, at the 20th week of culture, 8 Macrobrachium rosenbergii were taken from each group and boiled in boiling water for 3 minutes, then removed and cooled at room temperature to measure the color value of the cooked shrimp. The color difference value ΔE is calculated by the following formula: Where: L * is the brightness of the treatment group; is the brightness of the control group; a * is the redness of the treatment group, b is the redness of the control group; * is the yellowness of the treatment group, Yellowness of the control group.

6. The method for testing the effect of natural astaxanthin on the growth, body color and immunity of Macrobrachium rosenbergii as claimed in claim 2, characterized in that: The immune and antioxidant enzyme activity assays: The total superoxide dismutase (SOD), catalase (CAT), malondialdehyde (MDA), alkaline phosphatase (AKP), and lysozyme (LZM) activities in the hepatopancreas and hemolymph of shrimp at 10 and 20 weeks were determined according to the kit instructions of Nanjing Jiancheng Bioengineering Institute (Nanjing, China), and the effects of astaxanthin on immunity and antioxidant effects were evaluated; Determination of immune and molting gene expression levels: Total RNA from hepatopancreas was extracted by Trizol method, and then the integrity of RNA was evaluated by 1% agarose gel electrophoresis, and the concentration of RNA was detected by Nanodrop-2000 spectrophotometer. cDNA was synthesized according to the instructions of M-MLV reverse transcription kit. Real-time fluorescence quantitative PCR was performed using StepOnePlus Real-Tim PCR system to evaluate the expression levels of immune and molting-related genes in Macrobrachium rosenbergii after the addition of astaxanthin. The reaction system included 1 μL (50 ng / μL) cDNA, 10 μL 2×SYBRGreen Premix (Universal), 1 μL primer (10 pmol / μL) and 7 μL double distilled water, with a total system of 20 μL. The reaction procedure was as follows: pre-denaturation at 95°C for 30 min; 35 cycles of denaturation at 95°C for 40 s, annealing at 60°C for 45 s, and extension at 72°C for 10 min; data acquisition at 72°C for 10 min, and then melting curves were obtained at 95°C for 5 s, 60°C for 30 s, and 95°C for 15 s. Astaxanthin content determination: 5 g of muscle sample was extracted with acetonitrile, defatted and concentrated with n-hexane, and the astaxanthin content in the muscle was detected by high performance liquid chromatography.

7. The method for testing the effect of natural astaxanthin on the growth, body color and immunity of Macrobrachium rosenbergii as claimed in claim 2, characterized in that: Statistical analysis of the data: GraphPad Prism 9 software was used to analyze the differences in growth indicators, chromaticity values, astaxanthin content, immune and antioxidant and related gene expression level parameters of each group; data were expressed as "mean ± standard error", P < 0.05 indicated a significant difference, and P < 0.01 indicated an extremely significant difference.

8. A detection system for the effect of natural astaxanthin on the growth, body color and immunity of Macrobrachium rosenbergii, which implements the test method for the effect of natural astaxanthin on the growth, body color and immunity of Macrobrachium rosenbergii as described in any one of claims 1 to 7, characterized in that: The detection system for the effect of natural astaxanthin on the growth, body color and immunity of Macrobrachium rosenbergii comprises: A collection module, used for collecting samples; A calculation module, used for calculating growth indicators; The measurement module is used for body color measurement, immune and antioxidant enzyme activity measurement, immune and molting gene expression level measurement, and astaxanthin content measurement. The analysis module is used to perform differential analysis on growth indexes, chromaticity values, astaxanthin content, immunity and antioxidant and related gene expression level parameters of each group using GraphPadPrism 9 software.

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