A pearl gentian stone fish feed additive, a fish meal reduced feed, a low-cost breeding method and application

Abstract

The application provides a pearl ginseng grouper feed additive, a feed with reduced fish meal, a low-cost breeding method and application, and belongs to the technical field of water product feed additive development. The application provides that limonene combined with oxidized trimethylamine can effectively avoid the decline of the growth performance, liver damage and / or intestinal damage of pearl ginseng grouper caused by the reduction of fish meal in the feed, and the two have a synergistic effect. Based on this, a feed additive for pearl ginseng grouper, a feed with reduced fish meal and a low-cost breeding method for pearl ginseng grouper are provided, which can effectively reduce the use amount of fish meal, thereby reducing the feed cost and the breeding cost, and will not reduce the growth performance of pearl ginseng grouper, and will not cause liver damage and / or intestinal damage.

Description

Technical Field

[0001] This invention belongs to the field of aquatic feed additive development technology, specifically relating to a pearl grouper feed additive, a feed with reduced fishmeal content, a low-cost aquaculture method and its application. Background Technology

[0002] Pearl Giant Grouper is a type of saddle-banded pearl giant grouper ( Epinephelus lanceolatus (paternal parent) and Brown-spotted Pearl Giant Grouper ( Epinephelus fuscoguttatus The pearl grouper (a hybrid of the mother and father) has advantages such as rapid growth, delicious meat, and high market value. As a typical carnivorous fish, the pearl grouper primarily uses fishmeal as its protein source, and fishmeal constitutes a large proportion of its feed, accounting for a significant portion of feed costs. The increasing scale of aquaculture has also led to a substantial consumption of fishmeal in the formulated feed for pearl grouper. Currently, protein sources used as fishmeal substitutes mainly include plant protein sources, animal protein sources, and single-cell protein sources. Plant and animal proteins have become ideal alternatives to fishmeal due to their low price, abundant resources, and rich content of nutrients required by aquatic animals. However, plant and animal proteins also have some drawbacks, such as low digestibility, poor palatability, and amino acid deficiencies. Single-cell proteins have high protein content and a rich amino acid composition, but they are expensive and have low yields. Furthermore, increasing research has confirmed that protein substitution has a certain threshold; when the fishmeal substitution ratio exceeds a certain threshold, the growth performance of the pearl grouper will significantly decline, leading to adverse effects on liver and intestinal health. Therefore, developing green, healthy, sustainable, and nutritionally complete fishmeal alternatives is particularly important for the development of pearl grouper farming. Summary of the Invention

[0003] To address the problems existing in the prior art, the first objective of this invention is to provide the application of limonene combined with trimethylamine oxide in avoiding the decline in growth performance, liver damage and / or intestinal damage in pearl grouper, as well as in the preparation of pearl grouper feed with reduced fishmeal content.

[0004] A second objective of this invention is to provide a pearl grouper feed additive, a pearl grouper feed with reduced fishmeal content, and its application in low-cost pearl grouper farming.

[0005] A third objective of this invention is to provide a low-cost method for farming pearl grouper.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides the application of limonene combined with trimethylamine oxide in avoiding decreased growth performance, liver damage and / or intestinal damage in pearl grouper, which is caused by a reduction in fishmeal in the feed.

[0007] This invention provides the application of limonene combined with trimethylamine oxide in the preparation of pearl grouper feed with reduced fishmeal content.

[0008] Preferably, the mass ratio of limonene to trimethylamine oxide is (1~3):(1~3).

[0009] This invention provides a feed additive for pearl grouper, the feed additive being composed of limonene and trimethylamine oxide, wherein the mass ratio of limonene to trimethylamine oxide is (1~3):(1~3).

[0010] This invention provides a fishmeal-reduced pearl grouper feed, wherein the fishmeal content of the feed is reduced by 40wt% to 50wt%, and the feed includes 0.2wt% to 0.6wt% of the aforementioned feed additive.

[0011] Preferably, in the feed, chicken meal, Clostridium ethanol, methionine and lysine are used as protein sources to replace 40wt%~50wt% of fish meal.

[0012] Preferably, the feed is composed of the following components: red fish meal, chicken meal, Clostridium ethanol, soybean protein concentrate, soybean meal, flour, fish oil, soybean oil, soybean lecithin, microcrystalline cellulose, calcium dihydrogen phosphate, methionine, lysine, choline chloride, compound premix, antioxidant, limonene, and trimethylamine oxide.

[0013] Preferably, the feed, by weight parts, comprises the following components: 25-35 parts red fish meal, 15-20 parts chicken meal, 5-8 parts ethanol clostridium protein, 10-15 parts soybean protein concentrate, 5-8 parts soybean meal, 12-16 parts wheat flour, 4-6 parts fish oil, 1-2 parts soybean oil, 1-2 parts soybean lecithin, 0.5-1 part microcrystalline cellulose, 1-2 parts calcium dihydrogen phosphate, 0.01-0.03 parts methionine, 0.02-0.06 parts lysine, 0.4-0.6 parts choline chloride, 0.8-1.2 parts compound premix, 0.05-0.15 parts antioxidant, 0.1-0.3 parts limonene, and 0.1-0.3 parts trimethylamine oxide.

[0014] The present invention also provides the application of the above-mentioned feed additive or pearl grouper feed in any of the following: (1) Improve the species diversity and richness of the gut microbiota of pearl grouper; (2) Reshaping the abundance of functional genera of the gut microbiota of pearl grouper; (3) Low-cost farming of pearl grouper.

[0015] The present invention also provides a low-cost farming method for pearl grouper, comprising the following steps: feeding the pearl grouper with the above-mentioned pearl grouper feed.

[0016] Compared with the prior art, the beneficial effects of the technical solution of the present invention are as follows: This invention combines limonene and trimethylamine oxide, both of which have significant synergistic effects. When fishmeal is replaced or partially replaced by other protein sources, it can effectively prevent the decline in the growth performance of pearl grouper, increase the activity of antioxidant enzymes in the liver of pearl grouper, reduce the total cholesterol content in the liver, prevent damage to the liver and intestinal tissue structure, improve the species diversity and richness of the intestinal flora of pearl grouper, significantly change the overall community structure of the intestinal flora, and maintain the health level of the liver and intestine.

[0017] This invention can effectively reduce the amount of fishmeal used in pearl grouper farming, thereby reducing feed and farming costs, without reducing the growth performance of pearl grouper or causing liver and / or intestinal damage. It is of great significance to the healthy and sustainable development of the pearl grouper industry. Attached Figure Description

[0018] Figure 1 Growth performance of pearl grouper.

[0019] Figure 2 Results of the synergistic effect on the growth performance of pearl grouper.

[0020] Figure 3 Results of detection of antioxidant enzyme activity and biochemical indicators in the liver of pearl grouper.

[0021] Figure 4 HE staining results of the liver of pearl grouper.

[0022] Figure 5 HE staining results of intestinal tissue from pearl gentian grouper.

[0023] Figure 6 Electron microscopic observation results of pearl grouper sections.

[0024] Figure 7 Venn diagram analysis results of gut microbiota in pearl grouper.

[0025] Figure 8 Results of α-diversity analysis of gut microbiota in pearl grouper.

[0026] Figure 9Results of β-diversity analysis of gut microbiota in pearl grouper.

[0027] Figure 10 Stacked diagram of gut microbiota at the phylum level in pearl grouper.

[0028] Figure 11 Stacked diagram of gut microbiota at the species and genus level in pearl grouper. Detailed Implementation

[0029] This invention provides the application of limonene combined with trimethylamine oxide in avoiding decreased growth performance, liver damage and / or intestinal damage in pearl grouper, wherein the decreased growth performance, liver damage and / or intestinal damage in pearl grouper is caused by a reduction in fishmeal in the feed, and the mass ratio of limonene to trimethylamine oxide is (1~3):(1~3), preferably 1:3, 1:2, 1:1, 2:1 or 3:1.

[0030] This invention provides the application of limonene combined with trimethylamine oxide in the preparation of pearl grouper feed with reduced fish meal content. Preferably, the fish meal reduction is 40wt%~50wt%, including fish meal reduction of 41wt%, 42wt%, 43wt%, 44wt%, 45wt%, 46wt%, 47wt%, 48wt%, or 49wt%.

[0031] When fishmeal is replaced or partially replaced by other protein sources, the growth performance of pearl grouper declines significantly, and liver and intestinal health are affected. This invention combines limonene and trimethylamine oxide, both of which have significant synergistic effects. When fishmeal is replaced or partially replaced by other protein sources, this effectively prevents the decline in growth performance of pearl grouper, increases the activity of antioxidant enzymes in the liver, reduces total cholesterol content in the liver, avoids damage to liver and intestinal tissue structure, and maintains liver and intestinal health.

[0032] The gut microbiota plays a crucial role in digestion and absorption, supporting intestinal digestive activity and thus determining the growth status of pearl grouper. This invention combines limonene and trimethylamine oxide to increase the species diversity and richness of the gut microbiota in pearl grouper, significantly altering the overall community structure. At the phylum level, it changes the abundance of Pseudomonadota, Bacteroidota, and Bacillota, optimizing the phylum structure. At the genus level, it significantly reduces... Clostridium , Megasphaera The relative abundance of [something] increased significantly. Photobacterium , Turicibacter , Romboutsia , Staphylococcus , Cetobacterium , Vibrio The relative abundance of these bacteria is remodeled in terms of the abundance of specific functional genera.

[0033] This invention provides a feed additive for pearl grouper, comprising limonene and trimethylamine oxide, wherein the mass ratio of limonene to trimethylamine oxide is (1~3):(1~3), preferably 1:3, 1:2, 1:1, 2:1, or 3:1. The pearl grouper feed additive of this invention is preferably used in feeds where fishmeal is replaced or partially replaced by other protein sources. These other protein sources may include plant protein sources, animal protein sources, and single-cell protein sources, such as chicken meal, Clostridium ethanolis protein, methionine, and / or lysine. The partial replacement may involve replacing 40wt%~50wt% of fishmeal, such as 41wt%, 42wt%, 43wt%, 44wt%, 45wt%, 46wt%, 47wt%, 48wt%, or 49wt%.

[0034] This invention provides a reduced-fishmeal feed for pearl grouper, wherein the fishmeal content is reduced by 40wt% to 50wt%, and the feed includes 0.2wt% to 0.6wt% of the aforementioned feed additives. Preferably, the feed uses chicken meal, Clostridium ethanol, methionine, and lysine as protein sources to replace 40wt% to 50wt% of the fishmeal. The fishmeal replacement amount can be selected as 41wt%, 42wt%, 43wt%, 44wt%, 45wt%, 46wt%, 47wt%, 48wt%, or 49wt%. The chicken meal, Clostridium ethanol, methionine, and lysine are preferably formulated in a mass ratio of 15-20:5-8:0.01-0.03:0.02-0.06, more preferably in a ratio of 17.34:6.96:0.02:0.04.

[0035] The pearl grouper feed with reduced fish meal content according to the present invention is preferably composed of the following components: red fish meal, chicken meal, Clostridium ethanol, soybean protein concentrate, soybean meal, flour, fish oil, soybean oil, soybean lecithin, microcrystalline cellulose, calcium dihydrogen phosphate, methionine, lysine, choline chloride, compound premix, antioxidant, limonene, and trimethylamine oxide.

[0036] The preferred composition ratio of the reduced-fish-meat pearl grouper feed of the present invention (by mass parts) is as follows: 25-35 parts red fish meal, 15-20 parts chicken meal, 5-8 parts ethanol clostridium protein, 10-15 parts soybean protein concentrate, 5-8 parts soybean meal, 12-16 parts wheat flour, 4-6 parts fish oil, 1-2 parts soybean oil, 1-2 parts soybean lecithin, 0.5-1 part microcrystalline cellulose, 1-2 parts calcium dihydrogen phosphate, 0.01-0.03 parts methionine, 0.02-0.06 parts lysine, 0.4-0.6 parts choline chloride, 0.8-1.2 parts compound premix, 0.05-0.15 parts antioxidant, 0.1-0.3 parts limonene, and 0.1-0.3 parts trimethylamine oxide. More preferably, it contains 30.25 parts red fish meal, 17.34 parts chicken meal, 6.96 parts ethanol clostridium protein, 13 parts soy protein concentrate, 6 parts soybean meal, 14 parts flour, 5 parts fish oil, 1.53 parts soybean oil, 1.5 parts soy lecithin, 0.86 parts microcrystalline cellulose, 1.5 parts calcium dihydrogen phosphate, 0.02 parts methionine, 0.04 parts lysine, 0.5 parts choline chloride, 1 part compound premix, 0.1 parts antioxidant, 0.2 parts limonene, and 0.2 parts trimethylamine oxide.

[0037] In the pearl grouper feed with reduced fishmeal content described in this invention, the compound premix is ​​preferably a vitamin and mineral premix, and the antioxidant is preferably ethoxyquin.

[0038] The preparation method of the reduced-fishmeal pearl grouper feed of the present invention includes: all feed ingredients are pulverized by a pulverizer and passed through a 50-100 mesh sieve. Then, each feed ingredient is accurately weighed according to the feed formula, and all powders (red fishmeal, chicken meal, Clostridium ethanol, soybean protein concentrate, soybean meal, flour, microcrystalline cellulose, calcium dihydrogen phosphate, methionine, lysine, choline chloride, compound premix, antioxidant, limonene, and trimethylamine oxide) are mixed evenly using a step-by-step mixing method (choline chloride is added in water-soluble form). Weighed oils (fish oil, soybean oil, soybean lecithin) and 20-50 wt% purified water are added to the mixed powders. The powders and oils are manually mixed, and then all raw materials are passed through a 20-50 mesh sieve to prevent fat agglomeration and ensure uniform mixing. Finally, the mixed material is extruded into 2-5 mm pellets using a twin-screw extruder. The pearl grouper feed with reduced fishmeal content described in this invention is dried indoors (10~15℃) and then stored in a self-sealing bag at -20℃ for later use.

[0039] The pearl grouper feed with reduced fishmeal content described in this invention can significantly reduce feed costs. For example, fishmeal currently costs up to 21,500 yuan / ton. Based on a fishmeal content of 50wt% in each ton of feed formula, when 40wt% of fishmeal is replaced, the cost of fishmeal usage per ton of feed can be reduced by 20% × 21,500 = 4,300 yuan / ton.

[0040] The present invention also provides the application of the above-mentioned feed additives or pearl grouper feed in any of the following: (1) improving the species diversity and richness of the intestinal flora of pearl grouper; (2) reshaping the abundance of functional bacteria in the intestinal flora of pearl grouper; (3) low-cost farming of pearl grouper.

[0041] This invention also provides a low-cost method for farming pearl grouper, comprising the following steps: feeding the aforementioned pearl grouper feed. The farming method described in this invention can significantly reduce the farming cost of pearl grouper by approximately 20%.

[0042] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0043] In a specific embodiment of the present invention, the compound premix refers to a vitamin and mineral premix (per kg of feed) comprising the following: Vitamin A acetate, 675,000 IU; Vitamin D3, 180,000 IU; dl-α-tocopheryl acetate, 6 g; mefenamic acid, 1.2 g; thiamine nitrate, 0.9 g; riboflavin, 1.35 g; pyridoxine hydrochloride, 1.05 g; cobalt cyanoacrylate, 0.0075 g; D-calcium pantothenate, 4.5 g; nicotinamide, 6.75 g; folic acid, 0.375 g; D-biotin, 0.015 g; L-ascorbic acid-2-phosphate, 15 g; inositol, 10 g; iron, 20 g; zinc, 9.6 g; manganese, 6.4 g; copper, 0.6 g; cobalt, 0.08 g; selenium, 0.04 g; iodine, 0.08 g. The antioxidant is ethoxyquin. Limonene and trimethylamine oxide were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0044] Unless otherwise specified, the following embodiments are all conventional methods.

[0045] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0046] Example 1 A feed additive for pearl grouper: obtained by mixing limonene and trimethylamine oxide in a mass ratio of 1:1.

[0047] Example 2 A feed additive for pearl grouper: obtained by mixing limonene and trimethylamine oxide in a mass ratio of 1:3.

[0048] Example 3 A pearl grouper feed additive: obtained by mixing limonene and trimethylamine oxide in a mass ratio of 3:1.

[0049] Example 4 A reduced-feed pearl grouper feed (by weight percentage): red fish meal 30.25%, chicken meal 17.34%, ethanol clostridium protein 6.96%, soybean protein concentrate 13%, soybean meal 6%, wheat flour 14%, fish oil 5%, soybean oil 1.53%, soybean lecithin 1.5%, microcrystalline cellulose 0.86%, calcium dihydrogen phosphate 1.5%, methionine 0.02%, lysine 0.04%, choline chloride 0.5%, compound premix 1%, antioxidant 0.1%, limonene 0.2%, and trimethylamine oxide 0.2%. The resulting pearl grouper feed is designated MX1.

[0050] Preparation method: All feed ingredients were pulverized using a grinder and passed through a 60-mesh sieve. Then, each feed ingredient was accurately weighed according to the feed formula, and all powders were mixed evenly using a step-by-step mixing method (choline chloride was added in water-soluble form). Weighed oil and 30wt% purified water were added to the mixed powder. The powder and oil were manually mixed, and then all ingredients were passed through a 40-mesh sieve to prevent fat agglomeration and ensure even mixing. Finally, the mixed material was extruded into 2.5mm pellets using a twin-screw extruder. The experimental feed was dried indoors at 14℃ and stored in resealable bags at -20℃ until use.

[0051] Example 5 A reduced-feed pearl grouper feed (by weight): 25 parts red fish meal, 15 parts chicken meal, 5 parts ethanol clostridium protein, 10 parts soybean protein concentrate, 5 parts soybean meal, 12 parts wheat flour, 4 parts fish oil, 1 part soybean oil, 1 part soybean lecithin, 0.5 parts microcrystalline cellulose, 1 part calcium dihydrogen phosphate, 0.01 parts methionine, 0.02 parts lysine, 0.4 parts choline chloride, 0.8 parts compound premix, 0.05 parts antioxidant, 0.1 parts limonene, and 0.3 parts trimethylamine oxide.

[0052] Preparation method: Same as in Example 4.

[0053] Example 6 A reduced-feed pearl grouper feed (by weight): 35 parts red fish meal, 20 parts chicken meal, 8 parts ethanol clostridium protein, 15 parts soybean protein concentrate, 8 parts soybean meal, 16 parts wheat flour, 6 parts fish oil, 2 parts soybean oil, 2 parts soybean lecithin, 1 part microcrystalline cellulose, 2 parts calcium dihydrogen phosphate, 0.03 parts methionine, 0.06 parts lysine, 0.6 parts choline chloride, 1.2 parts compound premix, 0.15 parts antioxidant, 0.3 parts limonene, and 0.1 parts trimethylamine oxide.

[0054] Preparation method: Same as in Example 4.

[0055] Comparative Example 1 A pearl grouper feed (by weight percentage): 55% red fish meal, 0% chicken meal, 0% ethanol clostridium protein, 13% soybean protein concentrate, 6% soybean meal, 14% wheat flour, 5% fish oil, 1.6% soybean oil, 1.5% soybean lecithin, 0.8% microcrystalline cellulose, 1.5% calcium dihydrogen phosphate, 0% methionine, 0% lysine, 0.5% choline chloride, 1% compound premix, 0.1% antioxidant, 0% limonene, and 0% trimethylamine oxide. The resulting pearl grouper feed is denoted as FM.

[0056] Preparation method: Same as in Example 4.

[0057] Comparative Example 2 A reduced-feed pearl grouper feed (by weight percentage): red fish meal 30.25%, chicken meal 17.34%, ethanol clostridium protein 6.96%, soybean protein concentrate 13%, soybean meal 6%, wheat flour 14%, fish oil 5%, soybean oil 1.53%, soybean lecithin 1.5%, microcrystalline cellulose 1.26%, calcium dihydrogen phosphate 1.5%, methionine 0.02%, lysine 0.04%, choline chloride 0.5%, compound premix 1%, antioxidant 0.1%, limonene 0%, and trimethylamine oxide 0%. The resulting pearl grouper feed is denoted as 45FM.

[0058] Preparation method: Same as in Example 4.

[0059] Comparative Example 3 A reduced-feed pearl grouper feed (by weight percentage): red fish meal 30.25%, chicken meal 17.34%, ethanol clostridium protein 6.96%, soybean protein concentrate 13%, soybean meal 6%, wheat flour 14%, fish oil 5%, soybean oil 1.53%, soybean lecithin 1.5%, microcrystalline cellulose 1.06%, calcium dihydrogen phosphate 1.5%, methionine 0.02%, lysine 0.04%, choline chloride 0.5%, compound premix 1%, antioxidant 0.1%, limonene 0.2%, and trimethylamine oxide 0%. The resulting pearl grouper feed is denoted as N1.

[0060] Preparation method: Same as in Example 4.

[0061] Comparative Example 4 A reduced-feed pearl grouper feed (by weight percentage): red fish meal 30.25%, chicken meal 17.34%, ethanol clostridium protein 6.96%, soybean protein concentrate 13%, soybean meal 6%, wheat flour 14%, fish oil 5%, soybean oil 1.53%, soybean lecithin 1.5%, microcrystalline cellulose 0.86%, calcium dihydrogen phosphate 1.5%, methionine 0.02%, lysine 0.04%, choline chloride 0.5%, compound premix 1%, antioxidant 0.1%, limonene 0.4%, and trimethylamine oxide 0%. The resulting pearl grouper feed is denoted as N2.

[0062] Preparation method: Same as in Example 4.

[0063] Comparative Example 5 A reduced-feed pearl grouper feed (by weight percentage): red fish meal 30.25%, chicken meal 17.34%, ethanol clostridium protein 6.96%, soybean protein concentrate 13%, soybean meal 6%, wheat flour 14%, fish oil 5%, soybean oil 1.53%, soybean lecithin 1.5%, microcrystalline cellulose 1.06%, calcium dihydrogen phosphate 1.5%, methionine 0.02%, lysine 0.04%, choline chloride 0.5%, compound premix 1%, antioxidant 0.1%, limonene 0%, and trimethylamine oxide 0.2%. The resulting pearl grouper feed is denoted as T1.

[0064] Preparation method: Same as in Example 4.

[0065] Comparative Example 6 A reduced-feed pearl grouper feed (by weight percentage): red fish meal 30.25%, chicken meal 17.34%, ethanol clostridium protein 6.96%, soybean protein concentrate 13%, soybean meal 6%, wheat flour 14%, fish oil 5%, soybean oil 1.53%, soybean lecithin 1.5%, microcrystalline cellulose 0.86%, calcium dihydrogen phosphate 1.5%, methionine 0.02%, lysine 0.04%, choline chloride 0.5%, compound premix 1%, antioxidant 0.1%, limonene 0%, and trimethylamine oxide 0.4%. The resulting pearl grouper feed is denoted as T2.

[0066] Preparation method: Same as in Example 4.

[0067] Test case Pearl grouper farming experiments were conducted using the feed formulations of Example 4 and Comparative Examples 1-6, respectively.

[0068] In this experiment, all 7 groups of feed were of equal protein and fat content, with crude protein of approximately 50% and crude fat of approximately 12%.

[0069] This experiment consisted of 7 groups, with 3 replicates per group and 30 fish per replicate. Pearl grouper were fed with each group's diet for 8 weeks, twice a day, until the fish appeared to be satiated (most of the fish stopped competing for food), and the amount of feed was recorded.

[0070] After the culture experiment, the fish were fasted for 24 hours, weighed, and the number of surviving fish was recorded. Survival rate, weight gain rate, and specific growth rate were calculated. Liver and intestinal tissues were collected from the pearl grouper for liver enzyme activity assays, liver and intestinal section examination, and intestinal flora analysis. All values ​​are expressed as mean ± standard error (Mean ± SEM). One-way ANOVA was used. When there were significant differences between treatment groups (P < 0.05), Duncan's test was used for multiple comparison analysis.

[0071] Pearl Giant Grouper Growth Performance Figure 1 As shown, the results indicated that the growth performance of the 45FM group was significantly lower than that of the FM group. Furthermore, the feed conversion ratio (FCR) was highest in the 45FM group and significantly higher than that of the FM and N2 groups (P<0.05). Conversely, the MX1 group exhibited the highest growth performance and was significantly higher than that of the 45FM group (P<0.05). Compared to the 45FM group, while the addition of 0.2% or 0.4% limonene or trimethylamine oxide alone could improve the growth performance of the pearl grouper, the improvement was limited.

[0072] The synergistic effect of limonene (A) combined with trimethylamine oxide (B) on the growth performance of pearl grouper was evaluated. The synergistic index was calculated as follows: E A =(W N1 FBW -W 45FM FBW ) / W 45FM FBW E B =(W T1 FBW -W 45FM FBW ) / W 45FM FBW E 理论 =E A +E B E 实际 =(W MX1 FBW -W 45FM FBW ) / W 45FM FBW If E 实际 >E 理论 This indicates a synergistic effect between limonene and trimethylamine oxide. The results are as follows: Figure 2 As shown, the results indicate that the combined application of limonene and trimethylamine oxide significantly improved the growth effect and was significantly higher than the theoretical sum of the two when added alone (E-theory), which also indicates that there is a significant synergistic effect between limonene and trimethylamine oxide.

[0073] The results of the detection of antioxidant enzyme activity and biochemical indicators in the liver of pearl grouper are as follows: Figure 3As shown, the results indicated that the malondialdehyde (MDA) content in group T1 was significantly higher than that in group FM (P<0.05). In superoxide dismutase (SOD), the 45FM group was significantly lower than that in groups T2 and MX1 (P<0.05). Total antioxidant capacity was highest in group N1 and significantly higher than groups 45FM, N2, and T1 (P<0.05), with little difference from group MX1. The addition of limonene, trimethylamine oxide, or a combination thereof all reduced total liver cholesterol, with the FM group showing significantly higher levels than groups N1, N2, and T2 (P<0.05).

[0074] HE staining results of pearl grouper liver as follows Figure 4 As shown in the figure (scale bar is 100 μm), the results indicate that the tissue structure in the 45FM group was significantly damaged, manifested as disordered cell arrangement, increased intercellular spaces, loose local tissue structure, and uneven staining distribution, suggesting poor tissue morphological integrity in this group. Compared with the 45FM group, the tissue morphology of the N1 and N2 groups was improved, with relatively clear cell outlines, more uniform cell arrangement, and improved tissue structural integrity, indicating that limonene has a certain repairing effect on damaged tissue. The tissue structure of the T1 and T2 groups was also improved compared with the 45FM group, with more regular cell arrangement, reduced local intercellular spaces, and more uniform staining distribution, indicating that trimethylamine oxide treatment can alleviate tissue damage to a certain extent. In the MX1 group treated with a combination of limonene and trimethylamine oxide, the overall tissue structure was well maintained, with clear cell outlines, more regular cell arrangement, and significantly improved tissue morphological integrity compared with the 45FM group. These results indicate that the combined treatment of limonene and trimethylamine oxide can promote the recovery of damaged tissue structure and has a good tissue repair effect.

[0075] HE staining results of intestinal tissue of pearl grouper are as follows: Figure 5 As shown in the figure (scale bar is 200 μm), the results indicate that the intestinal villi in the FM group were long, slender, and neatly arranged, with no obvious inflammatory cell infiltration. In the 45FM group, the villi were shorter and blunter, with some irregular tips, and the lamina propria of the mucosa was widened with inflammatory cell infiltration, suggesting intestinal mucosal damage. Compared with the 45FM group, after the addition of limonene or trimethylamine oxide, the villi height and morphology of each gradient group gradually recovered, and the inflammatory cell infiltration gradually decreased. Among them, the mucosal structure of the MX1 group was closest to that of the control group, suggesting that this treatment has a significant protective effect on the intestinal mucosa.

[0076] Electron microscopy sections of pearl grouper from groups FM, 45FM, and MX1 were performed, and the results are as follows: Figure 6As shown in the figure (scale bar is 500 nm), the results indicate that the ultrastructure of intestinal epithelial cells in the FM group was intact, with a large number of mitochondria in the cytoplasm, regular morphology, and clear cristae structure, and no obvious swelling or vacuolation was observed. Compared with the FM group, the intestinal epithelial cells in the 45FM group showed obvious ultrastructural damage, significant swelling of mitochondria of varying sizes, disappearance or breakage of some cristae, and a lighter matrix; vacuolation and an increase in electron-dense bodies in the cytoplasm indicated significant cell damage and degenerative changes. Compared with the 45FM group, the mitochondrial swelling of intestinal epithelial cells in the MX1 group was significantly reduced, most mitochondria had regular morphology and relatively intact cristae structure, with only a few showing mild swelling; vacuolation and a decrease in the number of electron-dense bodies in the cytoplasm were observed, and the cell ultrastructure was relatively intact, suggesting that the combined use of limonene and trimethylamine oxide has a certain protective effect on the intestinal epithelial cells of pearl grouper.

[0077] The gut microbiota of pearl grouper from groups 45FM and MX1 were analyzed.

[0078] Venn diagram as shown Figure 7 As shown, the results revealed a total of 411 OTUs in the 45FM and MX1 groups, of which 346 were unique to the 45FM group and 939 were unique to the MX1 group. These results suggest a certain amount of shared flora between the two groups, but the MX1 group possesses more unique OTUs, indicating a potentially richer phylogenetic profile.

[0079] Analysis of α-diversity of gut microbiota in pearl grouper, such as Figure 8 As shown, the results indicated no statistically significant differences in ACE, Chao1, Shannon, and Simpson indices, but the MX1 group had higher species diversity and richness than the 45FM group.

[0080] Analysis of β-diversity of gut microbiota in pearl grouper, such as Figure 9 As shown, principal coordinate analysis (PCoA) based on Bray-Curtis distance revealed significant differences in community structure between different groups of gut microbiota at the OTU / species level. The results indicated that PCo1 and PCo2 explained approximately 49.10% and 16.03% of the community differences, respectively. In the planar plot, the 45FM and MX1 groups were clearly separated along the PCo1 axis, exhibiting tight clustering with almost no overlap between groups, suggesting a significant difference in the overall community structure of the two gut microbiota groups, indicating a substantial alteration in β-diversity.

[0081] A stacked diagram of the gut microbiota of pearl grouper at the phylum level is shown below. Figure 10As shown in the figure, at the phylum level, the gut microbiota in both groups were mainly composed of dominant phyla such as Pseudomonadota, Bacillota, Bacteroidota, and Actinomycetota. Low-abundance phyla such as Cyanobacteriota, Fusobacteriota, Dependentiae, Planctomycetota, Acidobacteriota, and Patescibacteria were also observed. The relative abundance of Pseudomonadota and Bacteroidota in group MX1 differed from that in group 45FM, while the proportion of Bacillota also differed between the two groups, suggesting that intervention / state changes affected the structure of dominant phyla.

[0082] A stacked diagram of the gut microbiota of the pearl grouper at the species and genus level is shown below. Figure 11 As shown. At the genus level, Clostridium (Clostridium spp.) Photobacterium (Bacillus luminifera) Turicibacter genus, Megasphaera (Megacoccus) Romboutsia The main dominant genera are genus, and also include genus '[']', ... Staphylococcus (Staphylococcus) Roseateles genus, Cetobacterium (Cetobacterium) Vibrio (Vibrio) Sulfitobacter Classification. Compared to the 45FM group, in the MX1 group, Clostridium , Megasphaera The relative abundance decreased significantly. Photobacterium , Turicibacter , Romboutsia , Staphylococcus , Cetobacterium , Vibrio The relative abundance of the genera increased significantly, while the proportions of unclassified genera and “Other” categories differed, suggesting that the abundance of specific functional genera was remodeled in the MX1 group compared to the 45FM group.

[0083] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The application of limonene combined with trimethylamine oxide in preventing decreased growth performance, liver damage, and / or intestinal damage in pearl grouper, characterized in that, The decline in growth performance, liver damage, and / or intestinal damage in the pearl grouper were caused by a reduction in fishmeal in the feed.

2. Application of limonene combined with trimethylamine oxide in the preparation of pearl grouper feed with reduced fishmeal content.

3. The application according to claim 1 or 2, characterized in that, The mass ratio of limonene to trimethylamine oxide is (1~3):(1~3).

4. A feed additive for pearl grouper, characterized in that, The feed additive is composed of limonene and trimethylamine oxide, wherein the mass ratio of limonene to trimethylamine oxide is (1~3):(1~3).

5. A fishmeal-reduced feed for pearl grouper, characterized in that, The feed contains 40wt% to 50wt% less fishmeal and includes 0.2wt% to 0.6wt% of the feed additive described in claim 4.

6. The pearl grouper feed according to claim 5, characterized in that, In the feed, chicken meal, Clostridium ethanol, methionine and lysine are used as protein sources to replace 40wt%~50wt% of fish meal.

7. The pearl grouper feed according to claim 5, characterized in that, The feed consists of the following components: red fish meal, chicken meal, Clostridium ethanol, soybean protein concentrate, soybean meal, flour, fish oil, soybean oil, soybean lecithin, microcrystalline cellulose, calcium dihydrogen phosphate, methionine, lysine, choline chloride, compound premix, antioxidant, limonene, and trimethylamine oxide.

8. The pearl grouper feed according to claim 5, characterized in that, The feed, by weight, comprises the following components: 25-35 parts red fish meal, 15-20 parts chicken meal, 5-8 parts ethanol clostridium protein, 10-15 parts soybean protein concentrate, 5-8 parts soybean meal, 12-16 parts wheat flour, 4-6 parts fish oil, 1-2 parts soybean oil, 1-2 parts soybean lecithin, 0.5-1 part microcrystalline cellulose, 1-2 parts calcium dihydrogen phosphate, 0.01-0.03 parts methionine, 0.02-0.06 parts lysine, 0.4-0.6 parts choline chloride, 0.8-1.2 parts compound premix, 0.05-0.15 parts antioxidant, 0.1-0.3 parts limonene, and 0.1-0.3 parts trimethylamine oxide.

9. The use of the feed additive according to claim 4 or the pearl grouper feed according to any one of claims 5 to 8 in any of the following, characterized in that, (1) Improve the species diversity and richness of the gut microbiota of pearl grouper; (2) Reshaping the abundance of functional genera of the gut microbiota of pearl grouper; (3) Low-cost farming of pearl grouper.

10. A low-cost farming method for pearl grouper, characterized in that, Includes the following steps: Feed the pearl grouper feed as described in any one of claims 5 to 8.

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