Aquaculture feed additive for alleviating heat stress of aquatic animals, feed and preparation method thereof

Abstract

The application discloses an aquatic feed additive for relieving heat stress of aquatic animals, a feed and a preparation method thereof, and the aquatic feed additive comprises, in terms of weight fractions, 3-10 parts of yeast culture, 0.5-2 parts of dandelion extract, 0.5-2 parts of citrus peel extract, 1-3 parts of beer yeast hydrolysate and 1-3 parts of grape seed extract. The yeast culture and the various plant extracts are optimally combined and synergistically act, the comprehensive ability of the aquatic animals to resist heat stress is improved in multiple ways and multiple levels, the adverse effects such as survival rate reduction caused by heat stress are effectively relieved, and the production performance of the aquatic animals is significantly improved.

Description

Technical Field

[0001] This invention relates to the field of aquatic feed technology, and in particular to an aquatic feed additive, feed, and preparation method for alleviating heat stress in aquatic animals. Background Technology

[0002] The Chinese mitten crab (Eriocheirsinensis), an important economic crustacean, possesses unique physiological characteristics. During its growth, the crab undergoes multiple molts to adapt to its increasing body size. Studies have shown that heat stress affects the molting process. Some research has found that molting frequency may increase under high temperatures, possibly due to hormonal changes and accelerated metabolism caused by heat stress. However, frequent molting can also lead to excessive energy consumption, affecting the crab's growth and survival. Furthermore, crabs are territorial and aggressive, and fighting is likely to occur under high-density farming conditions. Heat stress may exacerbate this aggressive behavior, leading to injury or even death, thus impacting farming efficiency.

[0003] The gut is a unique functional organ in aquatic animals, not only responsible for digesting food and absorbing nutrients, but also serving as a mucosal barrier to prevent harmful substances such as bacteria and toxins from invading the body. It is an important innate immune barrier closely related to the health and growth performance of aquatic animals. Studies show that heat stress can cause intestinal damage and oxidative stress in aquatic animals, leading to pathogen invasion, inflammation, and disease. Furthermore, heat stress can alter the gut microbiota structure, resulting in a decrease in beneficial bacteria and an increase in harmful bacteria, further impacting the host's health. Therefore, maintaining intestinal integrity, enhancing antioxidant capacity, and regulating gut microbiota balance are important strategies for alleviating heat stress in the Chinese mitten crab. Summary of the Invention

[0004] In view of the problems existing in the prior art, the present invention provides an aquatic feed additive, feed and preparation method for alleviating heat stress in aquatic animals. Through the optimized combination and synergistic effect of yeast culture and various plant extracts, it improves the comprehensive ability of aquatic animals to resist heat stress through multiple pathways and levels, effectively alleviates the adverse effects of heat stress such as decreased survival rate, and significantly improves the production performance of aquatic animals.

[0005] In a first aspect, the present invention provides an aquatic feed additive, comprising, by weight: 3-10 parts of yeast culture (YC), 0.5-2 parts of dandelion extract, 0.5-2 parts of citrus peel extract, 1-3 parts of brewer's yeast hydrolysate, and 1-3 parts of grape seed extract.

[0006] Preferably, the aquatic feed additive comprises, by weight, 5-8 parts of yeast culture (YC), 0.5-1.5 parts of dandelion extract, 0.5-1.5 parts of citrus peel extract, 1-2 parts of brewer's yeast hydrolysate, and 1.5-2 parts of grape seed extract.

[0007] Preferably, the aquatic feed additive comprises, by weight, 4.5 parts of yeast culture (YC), 1 part of dandelion extract, 1.5 parts of citrus peel extract, 1 part of brewer's yeast hydrolysate, and 2.0 parts of grape seed extract.

[0008] Preferably, the aquatic feed additive comprises, by weight, 6.0 parts of yeast culture (YC), 0.5 parts of dandelion extract, 0.5 parts of citrus peel extract, 1.5 parts of brewer's yeast hydrolysate, and 1.5 parts of grape seed extract.

[0009] The yeast culture is prepared by fermentation of a mixture of Saccharomyces cerevisiae, soybean meal, glucose, and gluten matrix. It contains various active substances, such as β-glucan, mannan oligosaccharides, nucleotides, and functional proteins, which can improve animal immunity, gut health, and stress resistance. Dandelion extract, obtained from the roots, leaves, or whole plant of dandelion through water or ethanol extraction, contains inulin, chicoric acid, taraxasterol, triterpenoid saponins, and other active ingredients, exhibiting antioxidant, choleretic, and digestive-promoting pharmacological effects. Citrus peel extract, obtained through water extraction or supercritical CO2 extraction, mainly contains flavonoids such as limonene and hesperidin, possessing antioxidant and anti-inflammatory bioactivities. Grape seed extract, prepared by ethanol extraction, mainly consists of proanthocyanidins and other active ingredients. Phenolic compounds possess strong antioxidant capabilities. The active ingredients of the various components mentioned above are natural and safe, and when combined, they can produce multiple synergistic effects: on the one hand, the β-glucan, mannan oligosaccharides, and other immunopolysaccharides and bioactive peptides provided by yeast cultures can directly enhance the body's immunity and stress resistance. On the other hand, plant-derived antioxidants such as polyphenols and flavonoids can scavenge reactive oxygen free radicals generated by heat stress and maintain the body's redox balance. Inulin, taraxasterol, and other components can regulate digestive function and promote nutrient absorption. Peptides, amino acids, nucleotides, and other nutrients provide the necessary nutrition for cell repair and regeneration. The synergistic effect of multiple active ingredients comprehensively enhances the ability of aquatic animals to resist heat stress from multiple aspects such as antioxidation, immune regulation, intestinal protection, and nutrient supply.

[0010] Secondly, the present invention provides a formulated aquatic feed, comprising, by weight, 8-10 parts of the above-mentioned aquatic feed additive and 800-1000 parts of basic feed.

[0011] Preferably, the base feed, by weight percentage, comprises: 15% fishmeal, 25% soybean meal, 25% cottonseed meal, 2% fish oil, 1.9% soybean oil, 0.5% soybean lecithin oil, 0.5% cholesterol, 0.5% choline chloride, 0.1% tert-butyl-p-cresol, 12% pregelatinized starch, 4% vitamin premix, 2% mineral premix, 2% sodium alginate, 1% betaine, 1% calcium dihydrogen phosphate, and 7.5% cellulose.

[0012] Thirdly, the present invention provides a method for preparing the above-mentioned aquatic compound feed, which includes the following steps:

[0013] S1. Take all the raw materials of the above aquatic feed additives according to the weight proportions, grind all the raw materials, and sieve them through a 60-mesh sieve.

[0014] S2. Thoroughly mix all the sieved raw materials to obtain the aquatic feed additive.

[0015] S3. Take the basic feed and aquatic feed additives from the above-mentioned aquatic compound feed according to the weight proportions, and add deionized water at a ratio of 100-200mL / kg of basic feed, and mix thoroughly to obtain a feed mixture.

[0016] S4. Use a twin-screw extruder to make the feed mixture into pellets with a diameter of 2-3 mm;

[0017] S5. Dry the pelleted feed at room temperature;

[0018] S6. Sift the dried pelleted feed through sieves of 10, 14 and 16 mesh in sequence.

[0019] S7. Pack the sieved feed from step S6 into self-sealing bags and store them at -20℃.

[0020] Fourthly, the present invention provides an application of the above-mentioned aquatic compound feed in improving the heat stress resistance of aquatic animals.

[0021] Preferably, improving the heat stress resistance of aquatic animals includes one or more of the following: regulating the intestinal microbial community structure, improving intestinal antioxidant capacity, alleviating heat stress damage, and maintaining the integrity of the intestinal barrier.

[0022] Preferably, the aquatic animal includes the Chinese mitten crab (Eriocheirsinensis).

[0023] In summary, the compound aquatic feed additive and its formulated feed provided by this invention, through the optimized combination and synergistic effect of yeast culture and various plant extracts, improve the comprehensive ability of aquatic animals to resist heat stress through multiple pathways and levels. It can effectively alleviate the adverse effects of heat stress, such as decreased survival rates, and significantly improve the production performance of aquatic animals. This compound additive is convenient to use, fast-acting, broad-spectrum, and highly effective, making it easy to promote and apply. Furthermore, the raw materials used in this invention are natural, green, and safe; the process is simple and easy to implement; and the production cost is low. It provides an economical, safe, and effective solution for the aquaculture industry to cope with high-temperature heat waves, and has significant practical implications for maintaining aquatic health, improving industry efficiency, and ensuring market supply. Attached Figure Description

[0024] Figure 1 The figure shows the effect of the compound feed of the present invention on the weight gain rate, survival rate and molting frequency of heat-stressed Chinese mitten crab.

[0025] Figure 2 The figure shows the effect of the compound feed of the present invention on heat stress-related hormones in Chinese mitten crabs.

[0026] Figure 3 The figure shows the effect of the compound feed of the present invention on the fighting behavior of heat-stressed Chinese mitten crab.

[0027] Figure 4 This is a graph showing the effect of the compound feed of the present invention on the relative expression level of heat shock protein genes in heat-stressed Chinese mitten crab.

[0028] Figure 5 The figure shows the effect of the compound feed of the present invention on the antioxidant enzyme activity of heat-stressed Chinese mitten crab.

[0029] Figure 6 This is a diagram showing the effect of the formulated feed of the present invention on the intestinal tissue structure of heat-stressed Chinese mitten crab.

[0030] Figure 7 This diagram illustrates the effect of the formulated feed of the present invention on biomarkers of intestinal damage in heat-stressed Chinese mitten crab.

[0031] Figure 8 This figure shows the effect of the formulated feed of the present invention on the species richness of intestinal microorganisms in heat-stressed Chinese mitten crabs.

[0032] Figure 9 This diagram illustrates the effect of the formulated feed of the present invention on the intestinal microbial community structure of heat-stressed Chinese mitten crab. Detailed Implementation

[0033] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the examples in the specification.

[0034] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0035] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in different places in this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that is mutually exclusive with other embodiments.

[0036] Example:

[0037] I. Preparation of Experimental Feed

[0038] Control group feed, experimental group feed 1, and experimental group feed 2 were prepared separately. The control group feed was the basal feed, while experimental group feed 1 and experimental group feed 2 were both formulated aquatic feeds according to this invention, differing only in the proportion of aquatic feed additives. The formulations of the three feeds are shown in Table 1. Yeast culture (YC) was provided by Diamond V Biological & Fermentation Engineering Technologies (Shenzhen) Co., Ltd., and was prepared by fermentation of a mixture of Saccharomyces cerevisiae, soybean meal, glucose, and gluten substrate.

[0039] Table 1 Experimental feed formulation and its nutritional components

[0040] Raw materials (g / kg) control group feed Experimental group feed 1 Experimental group feed 2 fish meal 150 150 150 soybean meal 250 250 250 cottonseed meal 250 250 250 Yeast culture (YC) 0 4.5 6.0 fish oil 20 20 20 Soybean oil 19 19 19 Soy lecithin 5 5 5 cholesterol 5 5 5 choline chloride 5 5 5 tert-butyl-p-cresol 1 1 1 Pregelatinized starch 120 115 115 Vitamin premix 40 40 40 Mineral premix 20 20 20 Sodium alginate 20 20 20 betaine 10 10 10 calcium dihydrogen phosphate 10 10 10 Cellulose 75 70 70 Dandelion extract 0 1 0.5 Citrus peel extract 0 1.5 0.5 brewer's yeast hydrolysate 0 1 1.5 Grape seed extract 0 2 1.5

[0041] In Table 1, the vitamin premix and mineral premix are both existing technologies, and their raw material ratios are sufficient to meet the growth requirements of the Chinese mitten crab.

[0042] The control group feed, experimental group feed 1, and experimental group feed 2 were dried at 105℃ until their mass was constant. Changes in feed moisture content were analyzed, and the crude protein content was determined using the Kjeldahl method. Crude fat was extracted using petroleum ether extraction. Simultaneously, the feed powder samples were thoroughly carbonized in a carbonization furnace and then calcined in a crucible furnace at 550℃ for at least 6 hours to determine the ash content. The moisture, crude protein, crude fat, and ash content results for the three feeds are shown in Table 2.

[0043] Table 2. Feed moisture and dry-basis composition results (%)

[0044] name Moisture Ash crude protein Crude fat control group feed 9.02 8.07 36.52 7.08 Experimental group feed 1 9.08 8.12 36.68 7.11 Experimental group feed 2 9.09 8.15 36.80 7.12

[0045] It can be seen that the crude protein and crude fat content of the three feeds did not change significantly, and all of them met the nutritional requirements for the normal growth and development of Chinese mitten crab.

[0046] II. Experimental Design and Sample Collection

[0047] Chinese mitten crabs (Eriocheir sinensis) were purchased and acclimatized in a laboratory for two weeks. During this period, the water temperature was maintained at 24.0±0.2℃, and the dissolved oxygen level was maintained at 6.8±0.2mg / L. After the acclimatization period, 720 healthy crabs (weighing 2.82±0.01g) were divided into four groups and placed in corresponding rearing tanks: control group 1, control group 2, experimental group 1, and experimental group 2, with 180 crabs in each group and 6 replicates per group (n=6). The dimensions of the rearing tanks were 87×65.3×61.5cm, and the bottom of each tank was equipped with arched tiles and corrugated plastic pipes as hiding places for the Chinese mitten crabs.

[0048] The water temperature in control group 1 was maintained at 24.0±0.2℃, and the animals were fed control group feed. The water temperature in control group 2 was maintained at 30.0±0.2℃, and the animals were fed control group feed. The water temperature in experimental groups 1 and 2 was maintained at 30.0±0.2℃, and the animals were fed experimental group feed 1 and experimental group feed 2, respectively. Other conditions, such as pH range of 7.3-8.4, ammonia nitrogen concentration below 0.05mg / L, and dissolved oxygen concentration above 7.0mg / L, were the same for all four groups.

[0049] The crabs in each group were cultured for 42 days, and during this period, each group was fed three times a day (once at 08:00, 14:00, and 20:00), with each feeding amount being 4-6% of the crab seedlings' body weight. Simultaneously, normal daily management was carried out during the culture period, including siphoning uneaten feed and feces, removing dead individuals, and recording molting frequency.

[0050] III. Behavioral Testing

[0051] After the rearing experiment, the survival rate, weight, and weight gain rate of each group of crab larvae were evaluated. Half of the crabs in each tank were used for behavioral testing, while the rest were anesthetized for subsequent dissection and sampling. Twenty crabs of similar size and sex (3-4 crabs per tank) were randomly selected from six parallel rearing tanks in each treatment group (Control Group 1, Control Group 2, Experimental Group 1, Experimental Group 2). The selected crabs were randomly paired into 10 pairs for testing. During testing, the paired crabs were simultaneously placed in identical test tanks, and their behavioral responses were observed and recorded, including fighting, fleeing, or no response. A higher proportion of fighting indicated stronger aggression. All test crabs were used only once.

[0052] IV. Biochemical Indicator Analysis

[0053] Crabs used for sampling were fasted for 24 hours before sampling. Ten crabs were then randomly selected from each tank and anesthetized by placing them on crushed ice. Hemolymph was carefully extracted from the base of the appendages of each anesthetized crab using a 1mL syringe and stored in 1.5mL centrifuge tubes, which were then stored overnight at 4°C.

[0054] After hemolymph collection, the 10 crabs were immediately dissected. The hepatopancreas of each crab was removed with forceps. The hepatopancreas tissue of 4 crabs was immediately placed in liquid nitrogen and stored at -80°C for subsequent gene expression analysis. The hepatopancreas tissue of the remaining 6 crabs was homogenized into one sample and a homogenate (i.e., 10% tissue homogenate) was prepared at a ratio of tissue mass (g) to 0.85% physiological saline volume (mL). The homogenate was placed in a water bath at 0°C and then centrifuged at 1500g for 15 minutes. The supernatant was collected and stored at -80°C for the determination of malondialdehyde content, glutathione peroxidase, and superoxide dismutase activities.

[0055] Simultaneously, intestinal tissue was extracted, and the intestinal tissues of 10 crabs from the same tank were homogenized into one sample. Intestinal tissue homogenate was prepared using the same method as for hepatopancreatic tissue homogenate, and centrifuged at 1500g for 15 minutes. The supernatant was collected and stored at -80℃ for histamine content determination. Meanwhile, four crabs were randomly selected from each tank, and their intestinal tissues were preserved in Bouin's solution for subsequent histological analysis. The intestinal contents were carefully collected, and the intestinal contents of the four crabs from the same tank were mixed into one sample, placed in a sterile 1.5mL centrifuge tube, immediately flash-frozen in liquid nitrogen, and then transferred to a -80℃ freezer for subsequent intestinal flora analysis.

[0056] Overnight hemolymph samples were thoroughly crushed with a syringe needle until no obvious clots were found. They were then centrifuged at 7000 rpm and 4°C for 10 minutes. The supernatant was carefully aspirated, aliquoted into 0.2 mL centrifuge tubes, and stored at -80°C.

[0057] V. Gene Expression Analysis

[0058] Total RNA was extracted from preserved hepatocellular and pancreatic tissue samples using TRIzol reagent. RNA was reverse transcribed into cDNA using a reverse transcription kit. The reaction volume was 20 μL, including 10 μL of 2×SYBR Green PCR Master Mix, 0.8 μL of forward and reverse primers (10 μM), 1 μL of cDNA template, and 8.2 μL of nuclease-free water. qRT-PCR was performed using a real-time quantitative PCR system, with β-actin as an internal control gene, to compare the relative mRNA expression levels between groups.

[0059] All data are expressed as mean ± standard error (SEM). Statistical analysis was performed using SPSS 20.0 software. One-way ANOVA was used to determine significant differences among groups at 30 ± 0.2℃ under the same water temperature conditions. If one-way ANOVA indicated a significant difference, Duncan's multiple comparison method was used to assess the significance between groups. Furthermore, t-tests were used to compare the differences between control group 1 and control group 2, and between experimental group 1 and experimental group 2. A p-value < 0.05 was considered statistically significant. Values ​​with different superscripts in the same row indicate significant differences; an asterisk indicates the significance level (* indicates p < 0.05; ** indicates p < 0.001). <p<0.01)。

[0060] VI. Gut microbiota sequencing and analysis

[0061] use Genomic DNA was extracted from intestinal contents samples using the Soil DNA Kit (Omega Biotek, USA). The V3-V4 regions of the bacterial 16S rRNA gene were amplified and sequenced using the Illumina platform. OTUs were defined using UPARSE software with a 97% similarity threshold, and species were classified using the Silva 138 database via RDP Classifier. α and β diversity indices were calculated using QIIME.

[0062] VII. Results Analysis

[0063] 1. Effects of formulated aquatic feed on weight gain, survival rate, and molting frequency of heat-stressed Chinese mitten crab.

[0064] Depend on Figure 1It can be concluded that, compared with control group 1, the weight gain rate of Chinese mitten crabs in control group 2 was significantly increased after being fed the basic feed at a high temperature of 30℃, but the survival rate was reduced. When the aquatic compound feed of the present invention was used, the survival rate of Chinese mitten crabs in experimental groups 1 and 2 was significantly improved, while the weight gain rate remained unchanged. At the same time, the molting rate of crabs in experimental groups 1 and 2 was significantly reduced. This indicates that the yeast culture in the compound feed of the present invention, combined with other natural extracts, can effectively alleviate the adverse effects of heat stress on the survival rate of Chinese mitten crabs through multiple mechanisms, while maintaining their growth performance. For example, it can maintain the weight gain rate at high temperatures and reduce the molting rate of crabs at high temperatures.

[0065] 2. Effects of formulated aquatic feed on molting-related hormones in heat-stressed Chinese mitten crab

[0066] Depend on Figure 2 It can be concluded that, compared with control group 1, the expression levels of crustacean hyperglycemic hormone and molting hormone mRNA in the hepatopancreas of *Eriocheir sinensis* in control group 2 were significantly upregulated, while the expression level of molting inhibitory hormone mRNA was significantly downregulated. When the aquatic feed additive of this invention was added to the basal feed at a high temperature of 30℃, the expression levels of crustacean hyperglycemic hormone and molting hormone mRNA in the hepatopancreas of *Eriocheir sinensis* were significantly reduced, and their expression levels were similar to those in control group 1, while the expression level of molting inhibitory hormone mRNA was significantly upregulated. This indicates that the synergistic effect of multiple functional components in the aquatic compound feed of this invention can effectively alleviate the effect of heat stress on the abnormal increase in molting in *Eriocheir sinensis* at the molecular level.

[0067] 3. Effects of formulated aquatic feed on fighting behavior in heat-stressed Chinese mitten crabs

[0068] Depend on Figure 3 It can be concluded that, compared with control group 1, the number of Chinese mitten crabs exhibiting fighting behavior increased in control group 2, while the number of those escaping did not change significantly. When the aquatic feed additive of this invention was added to the basic feed under a high temperature environment of 30℃, the number of Chinese mitten crabs exhibiting fighting behavior gradually decreased. This shows that the active ingredients produced by combining yeast culture with brewer's yeast hydrolysate, grape seed extract, dandelion extract, and citrus peel extract, such as the polypeptides and amino acids contained in brewer's yeast hydrolysate, can reduce fighting behavior in Chinese mitten crabs by improving central nervous system function, demonstrating a significant heat stress relief effect at the behavioral level.

[0069] 4. Effects of formulated aquatic feed on the relative expression levels of heat shock protein genes in heat-stressed Chinese mitten crab.

[0070] Depend on Figure 4It can be concluded that, compared with control group 1, the mRNA expression levels of heat shock protein genes hsp70 (i.e., "heat shock 70") and hsp90 (i.e., "heat shock 90") in the hepatopancreas of Chinese mitten crabs were significantly upregulated in control group 2. After adding the aquatic feed additive of the present invention to the basal diet, the mRNA expression levels of hsp70 and hsp90 in the hepatopancreas of Chinese mitten crabs were significantly reduced, and their expression levels were similar to those in control group 1. This result indicates that the aquatic feed additive of the present invention can significantly reduce the expression of heat shock protein genes at the molecular level. For example, it can indirectly regulate the expression of heat shock proteins by inhibiting related signaling pathways through flavonoids and polyphenols in citrus peel extract and grape seed extract, thereby alleviating the effects of heat stress on Chinese mitten crabs.

[0071] 5. Effects of formulated aquatic feed on the antioxidant enzyme activity of heat-stressed Chinese mitten crab

[0072] Depend on Figure 5 It can be concluded that, compared with control group 1, the antioxidant enzyme activity of Chinese mitten crabs in control group 2 was significantly reduced, specifically, the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-px) were decreased, while the content of malondialdehyde (MDA) was increased. However, after adding the aquatic feed additive of this invention to the basal feed, the SOD and GSH-px activities of Chinese mitten crabs in experimental groups 1 and 2 were significantly increased, while the MDA content was significantly reduced. This indicates that the aquatic feed additive of this invention, through the compounding of yeast culture with raw materials such as dandelion extract and grape seed extract, can effectively enhance the antioxidant capacity of Chinese mitten crabs under heat stress conditions and reduce the damage of oxidative stress to the body through the synergistic effect of multiple antioxidant components.

[0073] 6. Effects of formulated aquatic feed on intestinal tissue structure of heat-stressed Chinese mitten crab

[0074] Depend on Figure 6 It can be concluded that, compared with control group 1, the intestinal tissue of the Chinese mitten crabs in control group 2 showed significant damage, specifically manifested as shedding of the intestinal lining. However, when fed the aquatic compound feed of this invention at a high temperature of 30°C, the intestinal lining of the Chinese mitten crabs remained intact. This indicates that the aquatic feed additive of this invention can protect the intestinal tissue structure through active substances such as β-glucan provided by yeast culture and polypeptides provided by brewer's yeast hydrolysate, allowing the intestinal tissue of the Chinese mitten crabs to maintain its structural integrity even under heat stress conditions.

[0075] 7. Effects of formulated aquatic feed on intestinal damage biomarkers in heat-stressed Chinese mitten crab

[0076] Depend on Figure 7It can be concluded that, compared with control group 1, the diamine oxidase activity and lipopolysaccharide level in the serum of Chinese mitten crabs in control group 2 were significantly increased, and the histamine content in the intestine was also increased. All three substances are important biomarkers of intestinal damage. When fed the aquatic compound feed of this invention at a high temperature of 30℃, the diamine oxidase activity, lipopolysaccharide level, and histamine content all decreased, indicating that the aquatic feed additive in this invention also provides multiple protections to the intestine through various active substances, effectively reducing intestinal damage caused by heat stress and improving the intestinal physiological state of Chinese mitten crabs under high temperature conditions.

[0077] 8. Effects of formulated aquatic feed on gut microbial species richness in heat-stressed Chinese mitten crab

[0078] Depend on Figure 8 It can be seen that the species richness (ACE and Chao1 index) of the gut microbiota of Chinese mitten crabs in control group 2 was significantly lower than that in control group 1 (P < 0.05). When fed the aquatic compound feed of this invention at a high temperature of 30℃, the species richness of the gut microbiota of Chinese mitten crabs increased, indicating that the aquatic feed additives of this invention have a significant impact on microbial species richness. Among them, the prebiotics contained in brewer's yeast hydrolysate can play a key role in regulating gut microbiota diversity. In addition, flavonoids in citrus peel extract and inulin in dandelion extract also further optimize the gut microecology by providing selective carbon sources for beneficial bacteria, thereby indirectly enhancing the body's ability to resist heat stress.

[0079] 9. Effects of formulated aquatic feed on the gut microbiota structure of heat-stressed Chinese mitten crab

[0080] Figure 9 Principal component analysis (PCA) results showed significant differences between control group 2 and control group 1, and between experimental group 1 and experimental group 2, indicating that the aquatic feed additives of this invention can significantly affect the microbial community structure after feeding the aquatic compound feed of this invention at a high temperature of 30℃. Specifically, the flavonoids contained in citrus peel extract can selectively regulate specific bacterial groups, such as promoting the growth of beneficial bacteria like Lactobacillus and Bifidobacterium, and inhibiting potentially pathogenic bacteria, thereby improving the intestinal flora structure and enhancing the body's ability to adapt to heat stress. Simultaneously, the prebiotics provided by yeast culture and brewer's yeast hydrolysate also provide the necessary nutritional basis for the colonization and metabolism of these beneficial bacteria.

[0081] In summary, existing technologies have limited research on the application of multi-component compound additives in aquatic animals, especially those under heat stress. This invention employs a unique feed additive formulation, leveraging the effects of multiple active substances and the synergistic effects between different components to achieve a multi-pathway, multi-target synergistic effect. This effectively alleviates the adverse effects of heat stress on the Chinese mitten crab from multiple aspects, including growth performance, endocrine regulation, antioxidant function, and gut health. Furthermore, the feed additive components used in this invention are all natural, safe, and non-toxic raw materials, making it more environmentally friendly than conventional heat stress relief methods such as drug additives. This has significant implications for guiding production practices and promoting industrial development.

Claims

1. A feed additive for alleviating heat stress in Chinese mitten crabs, characterized in that, The feed additive, by weight, consists of the following components: 3-10 parts yeast culture, 0.5-2 parts dandelion extract, 0.5-2 parts citrus peel extract, 1-3 parts brewer's yeast hydrolysate, and 1-3 parts grape seed extract.

2. The feed additive as described in claim 1, characterized in that, The feed additive, by weight, consists of the following components: 5-8 parts yeast culture, 0.5-1.5 parts dandelion extract, 0.5-1.5 parts citrus peel extract, 1-2 parts brewer's yeast hydrolysate, and 1.5-2 parts grape seed extract.

3. The feed additive as described in claim 1, characterized in that, The feed additive, by weight, consists of the following components: 4.5 parts yeast culture, 1 part dandelion extract, 1.5 parts citrus peel extract, 1 part brewer's yeast hydrolysate, and 2 parts grape seed extract.

4. The feed additive as described in claim 1, characterized in that, The feed additive, by weight, consists of the following components: 6.0 parts yeast culture, 0.5 parts dandelion extract, 0.5 parts citrus peel extract, 1.5 parts brewer's yeast hydrolysate, and 1.5 parts grape seed extract.

5. A compound feed for aquatic life, characterized in that, Based on weight parts, it includes 8-10 parts of the feed additive as described in any one of claims 1-4 and 800-1000 parts of the basal feed.

6. The aquatic compound feed as described in claim 5, characterized in that, The basic feed, by weight percentage, consists of the following components: 15% fishmeal, 25% soybean meal, 25% cottonseed meal, 2% fish oil, 1.9% soybean oil, 0.5% soybean lecithin oil, 0.5% cholesterol, 0.5% choline chloride, 0.1% tert-butyl-p-cresol, 12% pregelatinized starch, 4% vitamin premix, 2% mineral premix, 2% sodium alginate, 1% betaine, 1% calcium dihydrogen phosphate, and 7.5% cellulose.

7. The method for preparing aquatic compound feed as described in claim 5, characterized in that, Includes the following steps: S1. Take all the raw materials of the feed additive described in any one of claims 1-4 by weight, grind all the raw materials, and sieve them through a 60-mesh sieve. S2. Thoroughly mix all the sieved raw materials to obtain the feed additive; S3. Take the basic feed and feed additives from the aquatic compound feed described in claim 5 by weight, and add deionized water at a ratio of 100-200 mL / kg of basic feed, and mix thoroughly to obtain a feed mixture. S4. Use a twin-screw extruder to make the feed mixture into pellets with a diameter of 2-3 mm; S5. Dry the pelleted feed; S6. Sift the dried pelleted feed through sieves of 10, 14 and 16 mesh in sequence. S7. Pack the sieved feed from step S6 into self-sealing bags and store them.

8. The use of the feed additive according to any one of claims 1-4 in the preparation of feed to improve the heat stress resistance of aquatic animals, wherein the aquatic animal is the Chinese mitten crab.

9. The application as described in claim 8, characterized in that, Enhancing the heat stress resistance of aquatic animals includes one or more of the following: regulating the gut microbiota structure, improving gut antioxidant capacity, alleviating heat stress damage, and maintaining the integrity of the intestinal barrier.

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