Exopalaemon carinicauda feed and preparation method and application thereof

Abstract

The application discloses a feed for Exopalaemon modestus, a preparation method and application thereof, and belongs to the field of aquaculture.The feed comprises bean cake, soybean concentrated protein, wheat flour, fish meal, cellulose, vitamin premix, mineral substance premix, a binder, phospholipid, choline, an attractant, soybean lecithin, fish oil and bean oil.The protein content in the feed is 35-45%.The feed and the feeding strategy can effectively improve the growth performance of the Exopalaemon modestus and stabilize the physiological state in the Exopalaemon modestus.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of aquaculture, and in particular, provides a feed for Exopalaemon carinicauda, a preparation method and application thereof. BACKGROUND

[0002] Exopalaemon carinicauda belongs to Custacea, Decapoda, Palaemonidae and Exopalaemon, and is mainly distributed in coastal waters in China, with the highest yield in the Yellow Sea, Bohai Sea and East China Sea. It is one of the three major indigenous economic shrimps in China and an important marine aquaculture object in the coastal areas north of Zhejiang Province. The current breeding methods and techniques are relatively backward. Breeders mainly refer to the breeding techniques of Chinese shrimp or South American white shrimp. At present, there is no specially developed compound feed, so the yield per unit area is not high. It is only used as an auxiliary or mixed breeding species and becomes part of pond culture, so its breeding status is low. At the same time, there are not many inputs for the development of related targeted breeding techniques and compound feeds at present, forming a vicious cycle and seriously affecting the development of Exopalaemon carinicauda breeding industry. Studies have shown that the breeding techniques and feeding factors transplanted from South American shrimp or Chinese shrimp are not suitable for Exopalaemon carinicauda. Unreasonable conditions can make the biological clock system of Exopalaemon carinicauda become disordered, thereby affecting growth and even promoting the development of adverse diseases. As a new emerging indigenous artificial breeding species in China, Exopalaemon carinicauda has led to a series of breeding problems due to lack of understanding or insufficient understanding of its biology, which cannot form a distinctive high-yield and high-efficiency breeding industry. SUMMARY

[0003] The technical problem solved by the present application is how to improve the growth performance of Exopalaemon carinicauda and balance the biological clock system of Exopalaemon carinicauda.

[0004] To solve the above problems, the first aspect of the present application provides a feed for improving the growth performance of Exopalaemon carinicauda. The feed comprises the following components in parts by mass: 10-15 parts of soybean meal, 15-25 parts of soybean protein concentrate, 15-25 parts of wheat flour, 14-25 parts of fish meal, 6-18 parts of cellulose, 1-4 parts of vitamin premix, 2-5 parts of mineral premix, 0.5-2 parts of adhesive, 0.5-1 part of phospholipid, 1-4 parts of choline, 0.1-1 part of attractant, 1-5 parts of soybean lecithin, 1-2 parts of fish oil, and 1-2 parts of soybean oil. The protein content in the feed is 35-45%.

[0005] Preferably, the vitamin premix comprises 0.5-2 parts of compound vitamin A and 0.5-2 parts of compound vitamin B.

[0006] Preferably, the phagostimulant is selected from one or more of squid meal, shrimp meal, chitin, dimethyl-beta-propanethine, betaine, trimethylamine oxide.

[0007] The second aspect of the present application provides a method for preparing the aforementioned feed, comprising the following steps:

[0008] S1, crushing: after mixing the soybean meal, soybean protein concentrate, wheat flour, fish meal, cellulose, vitamin premix, mineral premix, phospholipid, choline and phagostimulant, obtain the crushed material by ultrafine crushing;

[0009] S2, mixing: add soybean lecithin, fish oil and soybean oil to the crushed material, mix the mixture, then add the mixture to a mixer and mix with water to form a crude feed;

[0010] S3, granulation: use a granulator to make the crude feed into a granular feed, dry and cure to obtain a feed that is beneficial to the synchronization of the biological clock system of the Macrobrachium rosenbergii and the improvement of the growth performance of the Macrobrachium rosenbergii.

[0011] Preferably, in step S1, the particle size of the soybean meal, soybean protein concentrate, wheat flour, fish meal, cellulose, vitamin premix, mineral premix, phospholipid, choline and phagostimulant is 40-120 mesh.

[0012] Preferably, in step S3, the particle size of the granular feed is 0.5-3 mm, and the length is 0.5-3 mm.

[0013] Further, the third aspect of the present application provides a method for improving the synchronization of the biological clock system of the Macrobrachium rosenbergii, which uses the aforementioned feed that is beneficial to the growth performance of the Macrobrachium rosenbergii to feed the Macrobrachium rosenbergii, the feeding light period is 8-9L:15-16D, and the feeding time is selected from any one or more of 19:00, 22:00, 5:00, 0:00, 14:00 and 2:00 in order. In the preferred embodiment of the present application, the feeding time is preferably 2-3, and more preferably 3.

[0014] Further, the fourth aspect of the present application provides a method for screening and verifying the biological clock genes of the Macrobrachium rosenbergii, comprising the following steps:

[0015] Step a: sample the Macrobrachium rosenbergii under different feeding time treatments and perform transcriptome sequencing analysis, feed the aforementioned feed, and use qPCR technology to analyze the expression difference of the biological clock genes under different feeding time treatments to verify the accuracy of the genes and determine the core biological clock gene sequence of the Macrobrachium rosenbergii;

[0016] Step b: The changes in the expression rhythm of core biological clock genes are taken as the main changes in the biological clock system. The expression rhythm of biological clock genes in the central biological clock system and the expression rhythm in the peripheral biological clock system are used to judge the quality of the synchronization of the biological clock system. The quality of the synchronization of the biological clock system is used as the standard for judging the quality of feeding conditions.

[0017] Furthermore, the fifth aspect of the present invention provides a method for farming white shrimp, using the aforementioned feed that is beneficial to improving the growth performance of white shrimp. The farming method is as follows: salinity 22-25 ppt, temperature 23±1℃, photoperiod L:D = 8:16, light intensity 800-1300 lux, feeding time selected in sequence from any one or more of 19:00, 5:00, 22:00, 0:00, 14:00, and 2:00, feeding frequency 2-3 times / day, and feed weight 5% of the body weight of white shrimp.

[0018] The beneficial effects of this invention are as follows: The feed for white shrimp provided by this invention ensures the balance and stability of the white shrimp's biological clock system at the protein level. At the same time, the feeding strategy provided by this invention, which is conducive to maintaining the synchronization of the white shrimp's biological clock system and improving its growth performance, compares and analyzes the expression cycle, expression phase, and expression level of clock genes in the central and peripheral biological clock systems, as well as the expression of clock genes and metabolic genes, to determine the synergy between the central and peripheral clock systems and between clock genes and metabolic genes, and to determine the most suitable feeding strategy for improving the growth, development, and metabolic performance of white shrimp. Attached Figure Description

[0019] Figure 1 The figure shows the experimental results of the effect of different protein levels on the expression of circadian clock genes in the eyestalk of white shrimp in Example 1 of the present invention.

[0020] Figure 2 This is a graph showing the experimental results of the effect of different protein levels on the expression of circadian clock genes in the liver of white shrimp in Example 1 of the present invention;

[0021] Figure 3 This is a graph showing the experimental results of the effect of different photoperiods on the expression rhythm of biological clock genes in the eyestalk of white shrimp in Example 2 of the present invention;

[0022] Figure 4 This is a graph showing the experimental results of the effect of different photoperiods on the circadian rhythm of circadian clock genes in the liver of *Litopenaeus vannamei* in Example 2 of this invention.

[0023] Figure 5 This is a graph showing the experimental results of the effect of different feeding times on the expression rhythm of circadian clock genes in the eyestalks of white shrimp in Example 2 of the present invention;

[0024] Figure 6This is a graph showing the experimental results of the effect of different feeding times on the circadian rhythm of circadian clock genes in the liver of white shrimp in Example 2 of the present invention;

[0025] Figure 7 This is a graph showing the experimental results of the effect of different feeding frequencies on the expression of circadian clock genes in the eyestalks of white shrimp in Example 3 of the present invention;

[0026] Figure 8 This is a graph showing the experimental results of the effect of different feeding frequencies on the expression of biological clock genes in the liver of white shrimp in Example 3 of the present invention. Detailed Implementation

[0027] To make the above-mentioned objects, features, and advantages of the present invention more apparent and understandable, specific embodiments of the present invention are described in detail below. It should be noted that the following embodiments are only used to illustrate the implementation methods and typical parameters of the present invention, and are not intended to limit the parameter range described in the present invention. Reasonable variations derived therefrom are still within the protection scope of the claims of the present invention.

[0028] It should be noted that the endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0029] The feed preparation method provided in the specific embodiments of the present invention includes the following steps:

[0030] S1. Grinding: Take soybean meal, soybean protein concentrate, wheat flour, fish meal, cellulose, vitamin premix, mineral premix, phospholipids, choline and palatability enhancer, mix them and then grind them into fine powder.

[0031] S2. Mixing: Add soybean lecithin, fish oil and soybean oil to the crushed material, mix and then add the mixture to the mixer, add water and mix evenly to form roughage;

[0032] S3. Pelletizing: Using a pellet mill, roughage is processed into pellets, which are then dried and matured to produce feed that helps maintain the synchronicity of the biological clock system of white shrimp and improves their growth performance.

[0033] Example 1

[0034] Feed preparation

[0035] Prepare feed for white shrimp according to the feed formula in Table 1, where the values ​​in parentheses are set values.

[0036] Table 1 Feed Formulation

[0037]

[0038] Using the feed provided in this embodiment, the expression rhythm of circadian rhythm genes in the eyestalks and livers of *Litopenaeus vannamei* was investigated using real-time quantitative fluorescence technology. The specific implementation plan is as follows: 450 healthy, disease-free, uniformly sized, and vigorous *Litopenaeus vannamei* were randomly placed into 12 square glass aquariums and then placed in light incubators (temperature 26±1℃, salinity 23±1 ppt, light intensity 1000 lux). Three treatments were set up, labeled G1, G2, and G3 (corresponding to feed protein levels of 36%, 39%, and 42%, respectively). Specific growth rate (SGR), weight gain rate (WGR), and survival rate (SR) were calculated. Nutrient composition determination in muscle: moisture content was determined by atmospheric pressure drying, crude protein content by Kjeldahl nitrogen determination, crude fat content by Soxhlet extraction, and ash content by muffle furnace ignition. The Acro circadian rhythm analysis program was used to analyze raw data to determine whether the expression of target genes conformed to a cosine function, fitting the peak expression time points of target genes using cosine waves. One-way ANOVA was used to determine the statistical differences between different sampling times for each gene, and the Tukey test was used to determine the significance of differences between sample time points and the means of different sample sets. Growth performance and muscle composition data were statistically processed using SPSS 26.0 software, and one-way ANOVA was performed. Duncan's test was used to compare the significance of differences between groups, with P < 0.05 considered significant. Experimental data are expressed as mean ± standard deviation.

[0039] The formulas used for data calculation are as follows:

[0040] Specific growth rate (SGR, % / d) = 100 × [(lnWt - lnW0) / t]

[0041] Weight gain rate (WGR, %) = (Wt - W0) / W0 × 100

[0042] Survival rate (SR, %) = (Number of shrimp at the start of the experiment / Number of shrimp at the end of the experiment) × 100

[0043] In the formula, Wt is the final weight of the shrimp, W0 is the initial weight of the shrimp, and t is the culture time.

[0044] Experimental results are as follows Figures 1-2 As shown in Tables 2-4: Figure 1The figure shows the experimental results of the effects of different protein levels on the expression of circadian clock genes in the eyestalk of white shrimp. The presence of cosine waves indicates that the gene has significant diurnal rhythm, the thick black line represents the nighttime phase, and the horizontal axis is marked as the sampling time (ZT, Zeitgeber time).

[0045] Figure 2 Figure 1 shows the experimental results of the effects of different protein levels on the expression of circadian clock genes in the liver of white shrimp.

[0046] The four clock genes (Clock, Cry1, Tim, and Per2) analyzed in this experiment all exhibited significant diurnal rhythms in the eyestalks of *Litopenaeus vannamei*, showing a complete cycle of change within a 24-hour period. The timing of the peak expression phases of each gene was also largely consistent (P > 0.05), occurring in the late afternoon (ZT17). However, the expression levels of each gene were affected by dietary protein levels, resulting in significant differences in the trend curves of gene expression across treatments. Comparing the relative expression levels at the peak of each gene's expression phase, Clock expression in G1 was significantly lower than its expression in G2 and G3 (P < 0.05), while Per2 showed the opposite (P < 0.05). Cry1 and Tim exhibited the highest expression levels in treatment G3, significantly higher than their expression levels in the other two treatments. While each gene in the liver maintains a complete expression rhythm cycle within a 24-hour period, the timing of their respective peak phases varies significantly, as do the gene expression levels at the peak phase. Specifically, in G1, the peak phase of all four clock genes occurs around ZT21; in G2, Clock's peak phase occurs at ZT13, while the other three genes occur around ZT17; the variation is even greater in G3, with Tim's peak phase occurring at ZT1, and the remaining genes at ZT13. Looking at the expression levels of the four genes at the peak phase, only Cry1 and Tim show relatively similar expression levels across treatments, while Clock and Per2 show significant differences in expression levels across treatments. In the G1 and G3 treatments, the acrophase timing and relative expression levels of the four clock genes showed significant differences in the eyestalks and livers of *Litopenaeus vannamei*, with each index showing a significant difference (P<0.05). This indicates that these four genes showed no synchronicity between the central and peripheral circadian rhythm systems of *Litopenaeus vannamei* in the G1 and G3 treatments. In the G2 treatment, except for the Clock gene, which showed a significant difference in acrophase timing between the eyestalks and livers (P<0.05), the other genes did not show any differences (P>0.05). Regarding relative expression levels, Clock and Cry1 showed differences between the eyestalks and livers, while Tim and Per2 showed no difference. Therefore, in the G2 treatment, except for Clock, the other genes showed good synchronicity in both circadian rhythm systems.

[0047] Table 2 shows the experimental results of the effects of different protein levels on the growth of white shrimp. In the figure, different letters in each column indicate that there are significant differences between treatments (P < 0.05).

[0048] Table 3 shows the experimental results of the effects of different protein levels on the muscle composition of white shrimp. Different letters in each column indicate significant differences between treatments (P < 0.05).

[0049] Table 4 shows the results of the synchronous expression analysis of the four clock genes in the eye stalk and liver in each treatment. Different lowercase letters indicate that the relative expression level of the same gene in different parts of the same treatment is significantly different (P < 0.05); different uppercase letters indicate that the acromial time of the same gene in different parts of the same treatment is significantly different (P < 0.05).

[0050] The results above show that the feed provided in this embodiment can effectively improve the growth performance of white shrimp and enhance the stability of their biological clock system.

[0051] Table 2. Effects of different protein levels on the growth of whiteleg shrimp (n=10)

[0052]

[0053] Table 3. Effects of different protein levels on muscle composition of *Litopenaeus vannamei*.

[0054]

[0055] Table 4. Synchronization analysis of the expression of four clock genes in the eyestem and liver under each treatment.

[0056]

[0057] Example 2

[0058] This embodiment investigated the expression rhythms of circadian rhythm genes and some metabolic genes in the eyestalks and livers of *Litopenaeus vannamei* using real-time quantitative PCR technology, with different photoperiods and feeding times as variables. In this embodiment, the feed used was the feed prepared in Example 1. The specific implementation plan is as follows: This embodiment consists of three parts, with photoperiod and feeding time as the control conditions. The first part is the photoperiod embodiment, including two treatment groups (G1 and G2). In G1, the photoperiod is 12L:12D, with lights turned on at 6:00 AM and off at 6:00 PM; in G2, the photoperiod is 8L:16D, with lights turned on at 9:00 AM and off at 5:00 PM. Both groups were fed one hour after the lights were turned off. The second part is the feeding time embodiment, including six treatment groups labeled F5 (5:00), F10 (10:00), F14 (14:00), F19 (19:00), F22 (22:00), and F2 (2:00). Both experiments lasted 30 days. Feeding was stopped the day before sampling. The sampling start time for each treatment was set at 6:00 AM, and samples were taken every 4 hours (samples were labeled ZT6, ZT10, ZT14, ZT18, ZT22, and ZT2, respectively). Six shrimp were sampled each time (each sample was mixed with two shrimp × three replicates) and killed with an overdose of anesthetic. Their eyestalks and liver tissues were collected, stored in liquid nitrogen for 5 minutes, and then transferred to a -80°C freezer for subsequent analysis. The third part is the growth and rearing example. At the beginning, 10 shrimp were randomly selected from each rearing tank, and their initial body length and weight were measured. At the end of the experiment, 10 shrimp were again randomly selected from each rearing tank, and their final body length and weight were measured. Specific growth rate (SGR), weight gain rate (WGR), and survival rate (SR) were calculated. Nutritional composition determination in muscle: Moisture content was determined using the atmospheric pressure drying method, crude protein content using the Kjeldahl nitrogen determination method, crude fat content using the Soxhlet extraction method, and ash content using the muffle furnace ignition method. The Acro circadian rhythm analysis program was used to analyze the raw data to determine whether the expression of target genes conformed to a cosine function, and the time points of peak expression of target genes were fitted using cosine waves. One-way ANOVA was used to determine the statistical differences between different sampling times for each gene, and the Tukey test was used to determine the significance of differences between sample time points and the means of different sample sets. Growth performance and muscle composition data were statistically processed using SPSS 26.0 software, and one-way ANOVA was performed. Duncan's test was used to compare the significance of differences between groups, with P < 0.05 considered significant. Experimental data are expressed as mean ± standard deviation.

[0059] The formulas used for data calculation are as follows:

[0060] Specific growth rate (SGR, % / d) = 100 × [(lnWt - lnW0) / t]

[0061] Weight gain rate (WGR, %) = (Wt - W0) / W0 × 100

[0062] Survival rate (SR, %) = (Number of shrimp at the start of the experiment / Number of shrimp at the end of the experiment) × 100

[0063] In the formula, Wt is the final weight of the shrimp, W0 is the initial weight of the shrimp, and t is the culture time.

[0064] Experimental results are as follows Figures 3-6 As shown in Tables 5 and 6.

[0065] Figure 3 The figure shows the experimental results of the effect of different photoperiods on the expression rhythm of circadian clock genes in the eyestalk of white shrimp. The expression curves were generated by fitting with Acro software. The presence of cosine waves indicates significant diurnal rhythm. The thick black line represents the night phase.

[0066] Figure 4 The figure shows the experimental results of the effect of different photoperiods on the expression rhythm of circadian clock genes in the liver of white shrimp. The expression curves were generated by fitting with Acro software. The presence of cosine waves indicates significant diurnal rhythm. The thick black line represents the night phase.

[0067] Figure 5 The figure shows the experimental results of the effect of different feeding times on the expression rhythm of circadian clock genes in the eyestalk of white shrimp. The expression curves were generated by fitting with Acro software. The presence of cosine wave indicates a significant diurnal rhythm. The thick black line represents the night phase.

[0068] Figure 6 The figure shows the experimental results of the effect of different feeding times on the expression rhythm of circadian clock genes in the liver of white shrimp. The expression curves were generated by fitting with Acro software. The presence of cosine waves indicates a significant diurnal rhythm, and the thick black line represents the nighttime phase.

[0069] Table 5 shows the experimental results of the effects of different photoperiods / feeding times on the growth performance of white shrimp. In the table, different superscript letters in each column indicate significant differences between groups (P < 0.05).

[0070] Table 6 shows the experimental results of the effects of different photoperiods / feeding times on the composition of white shrimp. In this table, different superscript letters in each column indicate significant differences between groups (P < 0.05).

[0071] Table 5. Effects of different photoperiods / feeding times on the growth performance of white shrimp.

[0072]

[0073] Table 6. Effects of different photoperiods / feeding times on the composition of Litopenaeus vannamei.

[0074]

[0075] Analysis of circadian rhythm gene expression and its correlation with growth in *Litopenaeus vannamei* showed that different photoperiods and feeding times significantly affected the expression rhythms of circadian rhythm genes in the eyestalk and hepatomegaly. In the eyestalk, the acrosome of the Clock gene appeared near ZT19 under an 8L:16D photoperiod, which was nearly 4 hours later than under a 12L:12D photoperiod. Other circadian rhythm genes also showed similar changes in expression phase, indicating a significant correlation between the expression rhythms of circadian rhythm genes in the eyestalk and photoperiod. In the hepatomegaly, all circadian rhythm genes exhibited significant and highly similar rhythmic expression. Conversely, the expression rhythms of circadian rhythm genes in the eyestalk did not change with feeding time, while those in the hepatomegaly changed significantly with feeding time. The highest synchronicity of the circadian rhythm system was observed at 8L:16D and 19:00, resulting in the best growth performance and muscle composition, followed by 5:00 and 22:00. This indicates that different photoperiods or feeding times can affect the stable operation of the central and peripheral biological clock systems of white shrimp, causing them to separate and weaken or lose synchronization. This is because the photoperiod or feeding time is not suitable for the behavioral rhythm of white shrimp, and the synchronization of the biological clock system is disordered, thus reducing metabolic performance and growth performance.

[0076] Example 3

[0077] This embodiment investigated the expression rhythms of circadian rhythm genes and some metabolic genes in the eyestalks and livers of *Litopenaeus vannamei* using real-time quantitative PCR technology with different feeding frequencies as variables. The specific implementation plan is as follows: 600 healthy, disease-free, uniformly sized, and vigorous *Litopenaeus vannamei* were randomly placed in 12 square glass aquariums and then placed in a light incubator (temperature 26±1℃, salinity 23±1 ppt, light intensity 1000 lux). Four treatment groups were set up: Group A (fed once daily at 19:00), Group B (fed twice daily at 5:00 / 19:00), Group C (fed three times daily at 5:00 / 19:00 / 22:00), and Group D (fed four times daily at 5:00 / 14:00 / 19:00 / 22:00). Specific growth rate (SGR), weight gain rate (WGR), and survival rate (SR) were calculated. Nutritional composition of muscle was determined as follows: moisture content was measured using atmospheric pressure drying, crude protein content using the Kjeldahl method, crude fat content using Soxhlet extraction, and ash content using muffle furnace ignition. The Acro circadian rhythm analysis program was used to analyze the raw data to determine whether the expression of target genes conformed to a cosine function, and the time points of peak expression of target genes were fitted using cosine waves. One-way ANOVA was used to determine the statistical differences between different sampling times for each gene, and the Tukey test was used to determine the significance of differences between sample time points and the means of different sample sets. Growth performance and muscle composition data were statistically processed using SPSS 26.0 software, and one-way ANOVA was performed. Duncan's test was used to compare the significance of differences between groups, with P < 0.05 considered significant. Experimental data are expressed as mean ± standard deviation.

[0078] The formulas used for data calculation are as follows:

[0079] Specific growth rate (SGR, % / d) = 100 × [(lnWt - lnW0) / t]

[0080] Weight gain rate (WGR, %) = (Wt - W0) / W0 × 100

[0081] Survival rate (SR, %) = (Number of shrimp at the start of the experiment / Number of shrimp at the end of the experiment) × 100

[0082] In the formula, Wt is the final weight of the shrimp, W0 is the initial weight of the shrimp, and t is the culture time.

[0083] Experimental results are as follows Figures 7-8 As shown in Tables 7-9.

[0084] Figure 7 The figure shows the experimental results of the effect of different feeding frequencies on the expression of circadian clock genes in the eyestalks of white shrimp. The presence of cosine waves indicates that the gene has a significant diurnal rhythm, the thick black line represents the night phase, and the horizontal axis is marked as the sampling time (ZT, Zeitgeber time).

[0085] Figure 8 The figure shows the experimental results of the effect of different feeding frequencies on the expression of circadian clock genes in the liver of white shrimp. The presence of cosine waves indicates that the gene has a significant diurnal rhythm, the thick black line represents the night phase, and the horizontal axis is marked as the sampling time (ZT, Zeitgeber time).

[0086] Table 7 shows the results of the expression synchronization analysis of the four clock genes in the eye stalk and liver in each treatment. Different lowercase letters indicate that the expression parameters of the same gene in different parts of the same treatment are significantly different (P < 0.05).

[0087] Table 8 shows the experimental results of the effect of different feeding frequencies on the growth of white shrimp. Different lowercase letters in each column indicate significant differences between treatments (P < 0.05).

[0088] Table 9 shows the effects of different feeding frequencies on the muscle composition of white shrimp. In each column, different letters indicate significant differences between treatments (P < 0.05).

[0089] Table 7. Synchronization analysis of the expression of four clock genes in the eyestem and liver under each treatment.

[0090]

[0091] Table 8. Effects of different feeding frequencies on the growth of white shrimp.

[0092]

[0093] Table 9. Effects of different feeding frequencies on muscle composition of *Litopenaeus vannamei*.

[0094]

[0095] Analysis of the figures and tables in this embodiment shows that the four clock genes (Clock, Cry1, Tim, and Per2) analyzed in this example exhibited significant diurnal rhythms in the eyestalks of all four treatments of *Litopenaeus vannamei*, completing a full cycle within a 24-hour period. However, the expression of each gene differed under different feeding frequencies. Specifically, in treatments A and B, the Clock gene's apophase time appeared around ZT17, but the expression level of this gene differed significantly between the two treatments (P<0.05). In treatments C and D, the expression levels of the Clock gene were similar and not significantly different from those in treatment B, but the apophase time was earlier than in treatment B, with the earlier the apophase time occurring at higher feeding frequencies. The apophase expression of the other three genes did not change with the feeding frequency (P>0.05), but their expression levels were affected to some extent in each treatment. In the liver, all four clock genes also exhibited significant rhythmic changes, but unlike in the eyestalk, the expression cycles, acrophase occurrence times, and expression levels of each gene varied considerably, which is related to feeding frequency. As shown in the table, the synchronicity of gene expression in the eyestalk and liver was not entirely consistent across treatments A, B, and C. Differences existed in expression cycles, acrophase occurrence times, and relative expression levels. Overall, treatment C was slightly better, with most clock genes being synchronized except for the Clock gene, which was asynchronous. In treatment D, only the Cry1 gene showed good synchronicity in both tissues. Under different feeding frequencies, the specific growth rate (SGR) and weight gain (WGR) of *Litopenaeus vannamei* differed significantly among treatments. With increasing feeding frequency, both SGR and WGR significantly increased, reaching their maximum at a feeding frequency of 3 times / day (treatment C), consistent with the treatment group with the highest synchronicity of the biological clock system.

[0096] According to the experimental results of Examples 1 to 3, the following feeding strategies and feed protein levels (5:00, 19:00, 22:00, 3 times / day, 39% to 42%) improved the growth performance, muscle tissue protein content, and circadian rhythm homeostasis of white shrimp.

[0097] Unless otherwise defined, all terms, symbols, and other scientific terms used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In some instances, terms having a conventional meaning are defined herein for clarification or ease of reference, and such definitions should not be construed as indicating a significant difference from conventional understanding in the art. The technical methods described or referenced herein are generally well understood by those skilled in the art and employed by conventional methods. Unless otherwise stated, the use of commercially available reagents and instruments shall be performed according to the manufacturer's instructions and parameters.

[0098] While the disclosure is as stated above, its scope of protection is not limited thereto. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of this disclosure, and all such changes and modifications will fall within the protection scope of this invention.

Claims

1. A feed that improves the growth performance of whiteleg shrimp, characterized in that, The feed, by weight, comprises the following components: 10-15 parts soybean meal, 15-25 parts soybean protein concentrate, 15-25 parts wheat flour, 14-25 parts fish meal, 6-18 parts cellulose, 1-4 parts vitamin premix, 2-5 parts mineral premix, 0.5-2 parts binder, 0.5-1 part phospholipid, 1-4 parts choline, 0.1-1 part attractant, 1-5 parts soybean lecithin, 1-2 parts fish oil, and 1-2 parts soybean oil. The protein content of the feed is 39-42%. The attractant is selected from one or more of squid powder, shrimp powder, chitin, dimethyl-β-propionate thiobenzene, thiobetaine, and trimethylamine oxide. The vitamin premix includes 0.5-2 parts of compound vitamin A and 0.5-2 parts of compound vitamin B. The feed promotes the growth of white shrimp by regulating the acrophase time and gene expression level of the Tim and Per2 genes in the eyestalk.

2. A method for preparing the feed for improving the growth performance of whiteleg shrimp as described in claim 1, characterized in that, Includes the following steps: S1. Grinding: Take soybean meal, soybean protein concentrate, wheat flour, fish meal, cellulose, vitamin premix, mineral premix, phospholipids, choline and palatability enhancer, mix them and then grind them into fine powder. S2. Mixing: Add soybean lecithin, fish oil and soybean oil to the crushed material, mix and then add the mixture to the mixer, add water and mix evenly to form roughage; S3. Pelletizing: Using a pellet mill, roughage is processed into pellets, which are then dried and matured to produce feed that helps maintain the synchronicity of the biological clock system of white shrimp and improves their growth performance.

3. The method for preparing feed that improves the growth performance of white shrimp as described in claim 2, characterized in that, In step S1, the particle size of the soybean meal, soybean protein concentrate, wheat flour, fish meal, cellulose, vitamin premix, mineral premix, phospholipid, choline, and palatability enhancer is 40-120 mesh.

4. The method for preparing feed that improves the growth performance of white shrimp as described in claim 2, characterized in that, In step S3, the particle size of the pelleted feed is 0.5~3 mm and the length is 0.5~3 mm.

5. A method for improving the synchronization of the biological clock system of whiteleg shrimp, characterized in that, The white shrimp were fed with the feed described in claim 1 that is beneficial to improving the growth performance of the white shrimp. The photoperiod was 8-9L:15-16D and the feeding time was selected from any one or more of 19:00, 22:00, 5:00, 14:00 and 2:

00.

6. A method for farming white shrimp, characterized in that, The shrimp were fed using the feed described in claim 1 that is beneficial for improving their growth performance. The feeding method was as follows: salinity 22-25 ppt, temperature 23±1 ℃, photoperiod L:D=8:16, light intensity 800-1300 lux, feeding time selected from any two or three of 19:00, 5:00, 22:00, 14:00, and 2:00, and the feed weight was 5% of the shrimp's body weight.

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