Application of ginger extract as feed additive in improving growth performance and air exposure tolerance of procambarus clarkii

Abstract

The application provides application of a ginger extract as a feed additive in improving growth performance and air exposure tolerance of Procambarus clarkii and relates to the technical field of aquaculture. The inventors have found that the ginger extract as the feed additive added to feed can improve the growth performance, antioxidant capacity, immune defense capacity and air exposure tolerance of Procambarus clarkii, and reduce tissue damage and cell apoptosis of Procambarus clarkii induced by air exposure, and has good health cultivation and pre-transport nutrition strengthening application value.

Description

Technical Field

[0001] This invention relates to the field of aquaculture technology, and in particular to the application of ginger extract as a feed additive in improving the growth performance and air exposure tolerance of Procambarus clarkii. Background Technology

[0002] The red swamp crayfish (Procambarus clarkii) is an important freshwater aquaculture species in my country, characterized by high market demand, short breeding cycle, and strong adaptability. However, in actual breeding, harvesting, temporary holding, and transportation, red swamp crayfish are often affected by various environmental stressors, including air exposure, mechanical disturbance, overcrowding, temperature fluctuations, and water quality changes. Among these, air exposure is one of the most common stressors in the production and distribution of red swamp crayfish. Air exposure disrupts the normal respiration and metabolic processes of red swamp crayfish, causing an imbalance in redox homeostasis, promoting the accumulation of reactive oxygen species, and further inducing lipid peroxidation, tissue damage, apoptosis, and immune dysfunction, ultimately leading to decreased survival rate, reduced market quality, and increased economic losses. Therefore, improving the tolerance of red swamp crayfish to environmental stressors such as air exposure is an important technical direction for improving their breeding and transportation survival rates. Currently, the main technical means to improve the stress resistance of aquaculture animals include improving the breeding environment and transportation conditions, as well as using functional feed additives such as vitamins, minerals, probiotics, and immune enhancers. In recent years, plant-derived functional feed additives have received widespread attention in aquatic animal nutrition regulation due to their relatively natural sources, convenient application, green safety, and alignment with the trends of antibiotic reduction and replacement and healthy aquaculture. Ginger contains active ingredients such as gingerol, shogaol, volatile oils, and polyphenols, possessing potential functions such as antioxidant, anti-inflammatory, immunomodulatory, and tissue protection. Existing studies have shown that ginger or ginger extracts can improve growth, enhance antioxidant enzyme activity, and improve immunity in some fish and shrimp. However, research on the application of ginger extracts in the feed of Procambarus clarkii (red swamp crayfish) is still lacking. Existing technologies for healthy aquaculture and stress regulation of Procambarus clarkii still have the following shortcomings: 1) Insufficient nutritional regulation programs for air exposure stress. Procambarus clarkii inevitably experience out-of-water or semi-out-of-water environments during harvesting, transportation, and sales. Existing technologies mostly focus on transportation environment management, such as temperature control, humidity control, and reduction of mechanical damage, while there are few programs that improve the air exposure tolerance of Procambarus clarkii in advance through feed nutrition. 2) Conventional feed additives have limited functions and cannot simultaneously address both growth and stress resistance. Some feed additives can improve growth, while others can enhance immunity or antioxidant capacity. However, there are still limited additive programs that can simultaneously promote the growth of red swamp crayfish, improve antioxidant status, enhance immune indicators, and increase survival rate in air exposure.

[0003] In view of this, the present invention is hereby proposed. Summary of the Invention

[0004] The primary objective of this invention is to provide the application of ginger extract as a feed additive in improving the growth performance of Procambarus clarkii.

[0005] The second objective of this invention is to provide the application of ginger extract as a feed additive in improving the antioxidant capacity of Procambarus clarkii.

[0006] A third objective of this invention is to provide the application of ginger extract as a feed additive in enhancing the immune defense capabilities of red swamp crayfish.

[0007] A fourth objective of this invention is to provide the application of ginger extract as a feed additive in improving the air exposure tolerance of Procambarus clarkii.

[0008] The fifth objective of this invention is to provide the application of ginger extract as a feed additive in reducing tissue damage and apoptosis induced by air exposure in red swamp crayfish.

[0009] The sixth objective of this invention is to provide a feed for the red swamp crayfish.

[0010] The seventh objective of this invention is to provide the application of the above-mentioned Procambarus clarkii feed.

[0011] To achieve the above objectives, the following technical solution is adopted: In a first aspect, the present invention provides the application of ginger extract as a feed additive in improving the growth performance of Procambarus clarkii.

[0012] As a further technical solution, improving the growth performance of Procambarus clarkii includes increasing the final body weight, weight gain rate, growth rate, meat yield, or molting frequency of Procambarus clarkii.

[0013] Secondly, this invention provides the application of ginger extract as a feed additive in improving the antioxidant capacity of Procambarus clarkii.

[0014] Thirdly, this invention provides the application of ginger extract as a feed additive in improving the immune defense capabilities of Procambarus clarkii.

[0015] Fourthly, this invention provides the application of ginger extract as a feed additive in improving the air exposure tolerance of Procambarus clarkii.

[0016] Fifthly, the present invention provides the application of ginger extract as a feed additive in reducing tissue damage and apoptosis induced by air exposure in red swamp crayfish.

[0017] As a further technical solution, the ginger extract is added at 0.05%-0.80% of the feed mass; The ginger extract is a 70% vol ethanol extract of ginger.

[0018] In a sixth aspect, the present invention provides a feed for the red swamp crayfish, comprising ginger extract and a basic feed; The ginger extract is added at 0.05%-0.80% of the basic feed weight.

[0019] As a further technical solution, the ginger extract is a 70% vol ethanol extract of ginger.

[0020] In a seventh aspect, the present invention provides the application of the above-mentioned Procambarus clarkii feed in any of the following ae: a. Improve the growth performance of Procambarus clarkii; b. Enhance the antioxidant capacity of Procambarus clarkii; c. Enhance the immune defense capabilities of the red swamp crayfish; d. Improve the air exposure tolerance of Procambarus clarkii; e. Reduces tissue damage and apoptosis induced by air exposure in Procambarus clarkii.

[0021] Compared with the prior art, the present invention has the following beneficial effects: The inventors have discovered that adding ginger extract as a feed additive to feed can improve the growth performance, antioxidant capacity, immune defense capacity, and air exposure tolerance of red swamp crayfish after continuous feeding. It can also reduce tissue damage and cell apoptosis induced by air exposure in red swamp crayfish, demonstrating good application value for healthy aquaculture and pre-transport nutritional fortification. Attached Figure Description

[0022] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0023] Figure 1 : GE regulates the expression of growth and antioxidant-related genes; (AD) relative mRNA expression of growth-related genes; (EJ) relative mRNA expression of antioxidant-related genes; mRNA levels of target genes are expressed as follows: β-actin The internal references were normalized; data are expressed as mean ± standard error (n = 6); significance was tested using one-way ANOVA, followed by Duncan's multiple range test; different lowercase letters indicate significant differences between treatment groups. P <0.05); Figure 2 : GE can improve survival rate and modulate parameters related to oxidative stress after stress treatment; (A) Schematic diagram of experimental procedure; (B) Survival rate of GE0 and GE0.2 groups after 48 hours of air exposure; data are expressed as mean ± standard error (n = 3); (C) Effect of GE on oxidative stress-related parameters (including MDA, HSP70, HSP90, T-AOC, SOD, and CAT); data are expressed as mean ± standard error (n = 6); Student's t-test was used to compare survival rates; statistical significance among the four groups was assessed using one-way ANOVA, followed by Duncan's multiple range test;* P <0.05;** P <0.01; *** P <0.001; **** P <0.0001; Figure 3 : GE reduced tissue damage and apoptosis-related responses; (A) Histopathological assessment of hepatopancreatic and gill lesions based on H&E staining; Pathological notes: cell vacuolation and tissue destruction (red arrows), epithelial damage and epithelial cell migration (blue arrows); Scale bar = 200 µm; (BC) DAPI and TUNEL staining of the hepatopancreas; Scale bar = 75 µm; Data are mean ± standard error (n = 6); (DI) Relative expression levels of apoptosis-related genes; Data are expressed as mean ± standard error (n = 6); Significance was assessed using one-way ANOVA combined with Duncan's multiple range test;* P <0.05;** P <0.01; *** P <0.001; **** P <0.0001; Figure 4 : GE in feed was associated with enhanced Nrf2 / Keap1-related responses in air-exposed Procambarus clarkii; (AB) Quantitative analysis of Nrf2 nuclear translocation immunofluorescence and nuclear-cytoplasmic fluorescence ratio; Scale bar: 75 µm (n = 6); (CJ) Relative expression levels of Nrf2 / Keap1 pathway-related genes and downstream antioxidant / cytoprotective genes (n = 6); Significance was assessed using one-way ANOVA combined with Duncan's multiple range test;* P <0.05;** P <0.01; *** P <0.001; **** P <0.0001. Detailed Implementation

[0024] The embodiments and examples of the present invention will be described in detail below. However, those skilled in the art will understand that the following embodiments and examples are for illustrative purposes only and should not be considered as limiting the scope of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention. Unless otherwise specified, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0025] In a first aspect, the present invention provides the application of ginger extract as a feed additive in improving the growth performance of Procambarus clarkii.

[0026] The inventors' research has found that using ginger extract as a feed additive for red swamp crayfish can improve the crayfish's final body weight, weight gain rate, growth rate, meat yield, and molting frequency, and regulate... akt1 , mtor , 4ebp and s6k1 The expression of growth-related genes can improve the production performance of red swamp crayfish during the farming stage.

[0027] Secondly, this invention provides the application of ginger extract as a feed additive in improving the antioxidant capacity of Procambarus clarkii.

[0028] The inventors discovered that using ginger extract as a feed additive for Procambarus clarkii can promote Nrf2 nuclear translocation and improve... nrf2 Expression, reduction keap1 Express, and raise nqo1 , ho-1 , sqstm , gsts , cat and gpx The expression of downstream antioxidant and cell protection genes of Nrf2 indicates that it can enhance the tolerance of red swamp crayfish to air exposure stress by strengthening the antioxidant defense response mediated by Nrf2 / Keap1.

[0029] Thirdly, this invention provides the application of ginger extract as a feed additive in improving the immune defense capabilities of Procambarus clarkii.

[0030] The inventors discovered that using ginger extract as a feed additive for red swamp crayfish can increase the activity of antioxidant enzymes such as plasma SOD, reduce the accumulation of lipid peroxidation products such as MDA, and simultaneously increase the activity of non-specific immune-related enzymes such as ACP, AKP, and PO, thereby enhancing the basic health level and stress resistance of red swamp crayfish.

[0031] Fourthly, this invention provides the application of ginger extract as a feed additive in improving the air exposure tolerance of Procambarus clarkii.

[0032] The inventors' research found that using ginger extract as a feed additive for Procambarus clarkii can significantly improve the survival rate of Procambarus clarkii after 48 hours of air exposure stress, reduce air exposure-induced MDA accumulation and HSP70 levels, and has practical application value in reducing transportation losses and improving the survival rate of commercial shrimp.

[0033] Fifthly, the present invention provides the application of ginger extract as a feed additive in reducing tissue damage and apoptosis induced by air exposure in red swamp crayfish.

[0034] The inventors discovered that using ginger extract as a feed additive for red swamp crayfish can alleviate damage to the hepatopancreas and gill tissues caused by air exposure, reduce TUNEL-positive apoptosis signals in the hepatopancreas, and demonstrate reduced expression of the pro-apoptotic gene bax and increased expression of the anti-apoptotic gene bcl-2, proving its anti-stress protective effect at the tissue and cellular levels.

[0035] In some optional embodiments, the ginger extract is added at 0.05%-0.80% of the feed mass, preferably 0.10%-0.40%, and more preferably 0.20%. The ginger extract is a 70% vol ethanol extract of ginger. This invention does not impose specific limitations on the extraction conditions, but extraction is preferably carried out at room temperature. For example, the ginger extract can be prepared by pulverizing ginger, mixing it with 70% vol ethanol, extracting it at room temperature, performing solid-liquid separation, and drying the liquid portion to obtain the ginger extract.

[0036] In a sixth aspect, the present invention provides a feed for the red swamp crayfish, comprising ginger extract and a basic feed; The ginger extract is added at 0.05%-0.80% of the basic feed mass, preferably 0.10%-0.40%, and more preferably 0.20%.

[0037] The Procambarus clarkii feed provided by this invention uses ginger extract as a feed additive, and therefore possesses all the beneficial effects of ginger extract mentioned above.

[0038] This invention does not impose specific limitations on the composition of the basic feed; any feed for the red swamp crayfish well-known in the art can be used.

[0039] In some alternative embodiments, the ginger extract is a 70% vol ethanol extract of ginger.

[0040] In a seventh aspect, the present invention provides the application of the above-mentioned Procambarus clarkii feed in any of the following ae: a. Improve the growth performance of Procambarus clarkii; b. Enhance the antioxidant capacity of Procambarus clarkii; c. Enhance the immune defense capabilities of the red swamp crayfish; d. Improve the air exposure tolerance of Procambarus clarkii; e. Reduces tissue damage and apoptosis induced by air exposure in Procambarus clarkii.

[0041] The present invention will be further illustrated below with specific embodiments. However, it should be understood that these embodiments are merely for the purpose of more detailed illustration and should not be construed as limiting the present invention in any way.

[0042] Example 1 1.1 Materials and Methods 1.1.1 Ethical Statement All experimental procedures involving animals were reviewed and approved by the Ethics Committee of the Institute of Hydrobiology, Chinese Academy of Sciences (Approval No.: IHB / LL / 2025093).

[0043] 1.1.2 Experimental Feed The ginger extract used in this study was purchased from Nanjing Daosif Biotechnology Co., Ltd. (Nanjing, China; extraction solvent: 70% vol ethanol). The crude protein content of the basal feed was approximately 34%, and the crude fat content was approximately 5%. Ginger extract was added to the basal feed at concentrations of 0.00%, 0.05%, 0.10%, 0.20%, 0.40%, and 0.80% (w / w), respectively, and the corresponding treatment groups were named GE0, GE0.05, GE0.1, GE0.2, GE0.4, and GE0.8. All raw materials were pulverized and passed through a 60-mesh sieve and thoroughly mixed. The mixture was extruded into 2mm pellets using a single-screw extruder (SLP-45; Fisheries Machinery and Instrument Research Institute, Chinese Academy of Fishery Sciences, Shanghai, China). The pellets were conditioned at 95 °C for 20 min, dried at 60 °C, and stored at 4 °C for later use. The feed formulation and nutritional composition are shown in Table 1.

[0044] Table 1. Experimental feed formulation and nutrient composition (% dry matter)

[0045] Note: 1The vitamin premix contains 4 g of vitamin A, 0.02 g of vitamin D, 10 g of vitamin E, 10 g of vitamin K3, 10 g of vitamin B1, 10 g of vitamin B2, 20 g of vitamin B6, 40 g of niacin, 0.2 g of biotin, 20 g of calcium pantothenate, 0.5 g of folic acid, 0.01 g of vitamin B12, 20 g of vitamin C, and 400 g of inositol per kilogram, supplemented with microcrystalline cellulose to bring the total weight to 1 kg.

[0046] 2 The mineral salt premix contains 0.6 g of potassium iodate, 0.08 g of sodium selenite pentahydrate, 320 g of potassium dihydrogen phosphate, 200 g of magnesium sulfate, 20 g of manganese sulfate monohydrate, 2 g of copper chloride dihydrate, 60 g of zinc sulfate heptahydrate, 50 g of ferrous sulfate heptahydrate, 100 g of sodium chloride, and 2 g of cobalt chloride hexahydrate per kilogram, and is supplemented with microcrystalline cellulose to a total weight of 1 kg.

[0047] 1.1.3 Laboratory Animals and Breeding Conditions Juvenile red swamp crayfish used in the experiment were purchased from Jinghui Aquaculture Professional Cooperative in Hanchuan City (Hubei, China). Before the formal experiment, all crayfish were temporarily held under experimental conditions for one week. During this period, they were fed a basic feed twice daily.

[0048] After the initial holding period, 450 crayfish (initial weight 3.28 ± 0.03 g) were randomly assigned to 18 rearing tanks (1.2 m × 0.8 m; water depth 0.2 m), forming 6 treatment groups with 3 replicates per group and 25 crayfish per replicate. During the experiment, crayfish were fed experimental feed at 5% of their body weight twice daily for 8 weeks. The water temperature was maintained at 20.00 ± 0.49 °C, dissolved oxygen was maintained above 5.0 mg / L, total ammonia nitrogen was below 0.15 mg / L, and the photoperiod was 12 h light: 12 h darkness.

[0049] 1.1.4 Sample Collection After the culture experiment, the red swamp crayfish were fasted for 24 hours before sampling. The number and weight of crayfish in each culture tank were counted for calculating growth performance indicators. Three crayfish were randomly selected from each tank to measure body weight, muscle mass, and hepatopancreas weight. Hemolymph was collected using a sterile syringe containing sodium citrate anticoagulant; the anticoagulant formulation was: 4.8 g citric acid, 13.2 g sodium citrate, and 14.7 g glucose dissolved in 1 L of water, and sterilized by filtration. Hepatopancreas and gill tissue were obtained from dissection; a portion was fixed in 4% paraformaldehyde for histological analysis; the remaining tissue was immediately flash-frozen in liquid nitrogen for biochemical indicator determination and qPCR analysis.

[0050] 1.1.5 Growth performance determination After the culture experiment and sample collection, crayfish in the GE0 and GE0.2 groups were selected for air exposure stress experiments. The GE0.2 group was chosen as the representative addition level because it showed the most stable and consistent improvement in growth performance and plasma biochemical indicators during the culture experiment. Six crayfish were randomly selected from each culture box and transferred to a waterless culture box (temperature 20.8 °C; humidity 81%). Plastic shielding was provided inside the box to reduce cannibalism. After 48 hours of air exposure, samples were collected from the surviving individuals in each of the three culture boxes.

[0051] 1.1.6 Growth performance Growth performance was evaluated using final body weight (FBW), weight gain (WGR), specific growth rate (SGR), feed efficiency (FE), hepatic body index (HSI), meat yield (FC), and molting frequency.

[0052] 1.1.7 Plasma Collection and Biochemical Analysis Hemolymph samples were centrifuged at 3000 rpm for 10 min at 4 °C, and hemolymph plasma was collected. The activities of plasma alanine aminotransferase (ALT; C009-2-1) and aspartate aminotransferase (AST; C010-2-1) were measured as indicators of hepatopancreatic status. Malondialdehyde (MDA; A003-1-2), total antioxidant capacity (T-AOC; A015-2-1), superoxide dismutase (SOD; A001-3-2), and catalase (CAT; A007-1-1) were measured as indicators of oxidative stress. Acid phosphatase (ACP; A060-2-1), alkaline phosphatase (AKP; A059-2-2), and phenol oxidase (PO; H247-1-2) were measured as immune-related markers; HSP70 (H264-2-1) and HSP90 (H264-3-1) were also measured as stress-related protein markers. All assays were performed using commercial kits (Nanjing Jiancheng Biotechnology Institute, Nanjing, China) according to the manufacturer's instructions.

[0053] 1.1.8 Histological analysis and H&E staining Hepatopancreas and gill tissues were fixed in 4% paraformaldehyde, dehydrated with graded ethanol, embedded in paraffin, and then sectioned into 4 μm sections using a rotary microtome (Leica Biosystems, Germany). After dewaxing and rehydration, the sections were stained with hematoxylin and eosin (H&E). The tissue morphology was observed using an optical microscope.

[0054] 1.1.9 TUNEL staining Apoptotic cells in the hepatopancreas were detected using the TUNEL BrightGreen Apoptosis Detection Kit (Vazyme, China). Paraffin sections were treated with proteinase K (20 μg / mL) and equilibration buffer, and then incubated with TdT reaction solution at 37 °C for 1 h. After washing with PBS, the cell nuclei were counterstained with DAPI (2 μg / mL) for 5 min under light-protected conditions. Sections were mounted with 20% glycerol and imaged using a confocal microscope (Leica TCS SP8). One representative section from each animal was selected for each biological replicate, and non-overlapping fields were analyzed. The TUNEL positive signal was quantified using ImageJ software.

[0055] 1.1.10 Immunofluorescence analysis Hepatopancreatic sections were permeabilized with 0.02% Triton X-100 for 10 min and blocked with 5% goat serum for 30 min. The sections were then incubated overnight at 4 °C with anti-Nrf2 primary antibody (1:500, A3577, ABclonal), followed by incubation at room temperature in the dark with goat anti-rabbit secondary antibody (1:1000, A0207, ABclonal). After washing with PBS, cell nuclei were counterstained with DAPI. Negative controls were treated concurrently with the experimental groups but without primary antibody incubation. Fluorescence signals were observed using a confocal microscope (Leica TCS SP8), and random images were taken from each section. In each image, regions of interest (ROIs) were manually delineated based on tissue morphology and the distribution of DAPI-positive cells, excluding blank areas, debris, and nonspecific edge signals. The Nrf2 nuclear / cytoplasmic fluorescence intensity ratio was quantified using ImageJ.

[0056] 1.1.11 Real-time quantitative PCR Total RNA was extracted from the hepatopancreas using the TransZol Up Plus RNA Kit (TransGen Biotech, China), and RNA concentration and purity were detected using a NanoDrop ND-2000 spectrophotometer (NanoDrop Technologies, USA). cDNA was synthesized using the HiScript III RT SuperMix for qPCR kit (Vazyme, China). qPCR was performed on a LightCycler 480 II system (Roche, Basel, Switzerland). The 20 μL reaction mixture consisted of 10 μL of 2× SYBR Green Pro Taq HS Premix (Accurate Biotechnology, China), 2 μL of cDNA, 0.4 μL each of forward and reverse primers, and nuclease-free water to a final volume of 20 μL. The reaction program was as follows: 95 °C pre-denaturation for 5 min; followed by 35 amplification cycles, each consisting of 95 °C denaturation for 5 s and 60 °C annealing for 30 s; melting curve analysis was then performed, and the mixture was cooled to 4 °C. Primer sequences are shown in Table 2. β-actin This gene was used as an internal reference. Primer amplification efficiency was determined using a standard curve, and relative expression levels were calculated using the Pfaffl method (Pfaffl, 2001). All qPCR reactions were technically replicated.

[0057] Table 2 qPCR primer sequences

[0058] 1.1.12 Statistical Analysis Statistical analysis was performed using GraphPad Prism 10 software. Data are expressed as mean ± standard error (SEM), where n represents the number of biologically independent samples or experimental replicates as indicated in the legend. Normality and homogeneity of variance were tested before analysis. Based on the legend, Student's t-test or one-way ANOVA combined with Duncan's multiple comparison test was used to evaluate statistical differences. P A value <0.05 indicates that the difference is statistically significant.

[0059] 1.2 Results 1.2.1 Effects of feed GE (ginger extract) on growth performance Growth performance and feed utilization related indicators are shown in Table 3. Compared with the control group (GE0), the FBW of the GE0.1, GE0.2, GE0.4 and GE0.8 groups was significantly increased ( P<0.05). WGR and SGR were significantly increased in the GE0.2, GE0.4 and GE0.8 groups, while FE was significantly increased only in the GE0.1 group ( P <0.05. FC was significantly increased in all GE-added groups ( P <0.05%. The molting frequency was significantly increased in the GE0.1, GE0.2 and GE0.4 groups ( P <0.05). Overall, GE0.2 and GE0.4 showed the most consistent improvement in growth performance.

[0060] Table 3. Growth performance of Procambarus clarkii after 56 days of feeding with ginger extract.

[0061] Note: Data are average ± SEM (n=3).

[0062] Initial body weight (IBW, g) = Initial gross weight / Initial decimal; Final weight (FBW, g) = Total final weight / Final weight; Survival rate (SR, %) = 100 × number of terminal tails / number of initial tails; Weight gain rate (WGR, %) = 100 × (final weight - initial weight) / initial weight; Specific growth rate (SGR, % / d) = 100 × [ln(final body weight) - ln(initial body weight)] / number of days; Feed efficiency (FE, %) = 100 × (final body weight - initial body weight) / feed intake; Hepatopancreatic index (HSI, %) = 100 × hepatopancreatic weight / final body weight; Meat yield (FC, %) = 100 × Abdominal muscle weight / Final body weight; Molting frequency = total number of molts / number of tails.

[0063] 1.2.2 Effects of dietary GE on plasma biochemical parameters There were no significant differences in plasma ALT and AST activities among the groups (Table 1-4); P >0.05). As the level of GE addition increased, the overall plasma MDA content decreased, with the lowest level observed in the GE 0.4 group (Table 1-4); P <0.05). Among the antioxidant indicators, the SOD activity in the GE0.2 group was significantly higher than that in the GE0 group, and the GE0.4 group also showed a similar increasing trend (Table 1-4; compared with GE0, GE0.2, P<0.05). T-AOC and CAT also showed an increasing trend, but the differences did not reach statistical significance. Immune-related indicators also responded to GE addition: ACP and PO activities were significantly increased in all GE addition groups, and AKP activity was significantly increased at 0.10%–0.40% addition levels, with the peak generally occurring in the GE 0.2 group (Table 4; P <0.05). Overall, GE0.2 showed a relatively balanced response in terms of growth, antioxidant and immune-related indicators.

[0064] Table 4. Plasma biochemical indicators of red swamp crayfish after 56 days of feeding with ginger extract.

[0065] Note: Data are mean ± SEM (n=6). Different letters in the same row indicate significant differences between treatments. P <0.05).

[0066] 1.2.3 Effects of dietary GE on the expression of growth and antioxidant-related genes After 56 days of rearing, the GE0.05, GE0.1, and GE0.2 groups akt1 Expression levels were significantly upregulated ( Figure 1 A in the middle; P <0.05). mtor The expression level was highest in the GE0.2 group ( Figure 1 B in the middle; P <0.05), and 4ebp Expression levels were significantly reduced in the GE0.4 and GE0.8 groups. Figure 1 C in the middle; P <0.05). s6k1 Expression levels increased with medium-dose GE supplementation, then decreased, but the difference did not reach statistical significance. Figure 1 D in the middle; P >0.05). The above transcriptional expression pattern is consistent with the growth response results.

[0067] nrf2 , ho-1 , mnSOD and cuznSOD The expression level of [the substance] generally increased at low doses of GE supplementation, but decreased at the highest supplementation level. Figure 1 (E–H in the control group). Specifically, compared with the control group, the GE0.05, GE0.1, and GE0.2 groups nrf2 Expression levels were significantly upregulated; ho-1 Expression levels were highest in the GE0.2 and GE0.4 groups. P <0.05). mnSOD Expression levels peaked in the GE0.2 group. cuznSODExpression levels were significantly increased in the GE0.2 group ( P <0.05). Conversely, keap1 Expression levels decreased with increasing GE levels, reaching the lowest level in the GE 0.4 group. Figure 1 The I in P <0.05), and cat The expression level was not significantly affected. Figure 1 J in the middle; P >0.05).

[0068] 1.2.4 GE in feed reduces air exposure-induced oxidative stress and tissue damage At the end of 48 hours of air exposure (AE) stress, the survival rate of the GE0.2 group was higher than that of the GE0 group. Figure 2 B in the middle; P <0.05%. Air exposure significantly increased HSP70 and HSP90 levels, indicating that the cellular stress response was activated ( Figure 2 D–E in the middle; P <0.05%. Compared with the GE0 group after air exposure, the addition of 0.20% GE reduced MDA accumulation and HSP70 levels ( Figure 2 C–D in the middle; P <0.05. There were no significant differences in T-AOC, SOD, and CAT activities between the two air exposure groups. Figure 2 F–H in; P >0.05), but the overall trend suggests that the antioxidant status may have been partially improved.

[0069] Representative H&E sections showed that air exposure resulted in more severe damage to the hepatopancreas and gills, while the GE-added group showed relatively milder damage. Figure 3 (A) Consistent with histological observations, TUNEL staining showed increased apoptotic signaling after air exposure, while dietary GE supplementation decreased apoptotic signaling. Figure 3 B–C in the middle; P <0.05).

[0070] Air exposure also increased the expression of several pro-apoptotic genes, including bax , caspase2 , caspase3 , p53 and atf2 ,and bcl-2 The opposite trend is observed. Figure 3 D–I in; P <0.05). Under air exposure conditions, the addition of 0.20% GE significantly reduced [the concentration of GE] compared to the AE+GE0 group. bax Express and elevate bcl-2 Express( Figure 3 D and I in the middle;P <0.05). These results indicate that the reduction of tissue damage is accompanied by a weakening of pro-apoptotic transcriptional signatures, mainly manifested in bax and bcl-2 Regarding changes in expression: AE+GE0.2 group caspase2 , caspase3 , p53 and atf2 The expression level was lower than that of the AE+GE0 group, but the difference was not statistically significant.

[0071] 1.2.5 Nrf2 / Keap1 Correlation Response under GE-Regulated Air Exposure Conditions in Feed Immunofluorescence analysis showed that, under both basal and air-exposed conditions, dietary GE supplementation increased the Nrf2 nuclear / cytoplasmic fluorescence intensity ratio; the ratio was significantly higher in the AE+GE0.2 group than in the AE+GE0 group. Figure 4 A–B in the middle; P <0.05). At the transcriptional level, nrf2 The expression level was highest in the AE+GE0.2 group, while keap1 The expression level was significantly lower than that of the AE+GE0 group ( Figure 4 C–D in the middle; P <0.05). Air exposure increased significantly. nqo1 , gsts , cat and gpx mRNA expression. Compared with the AE+GE0 group, the AE+GE0.2 group... nqo1 , ho-1 , sqstm , gsts , cat and gpx The expression level also increased further. Figure 4 E–J in the middle; P <0.05). These results suggest that air exposure induces a compensatory Nrf2 / Keap1-related transcriptional response, and dietary GE further enhances this pathway response in surviving Procambarus clarkii.

[0072] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Application of ginger extract as a feed additive in improving the growth performance of Procambarus clarkii.

2. The application according to claim 1, characterized in that, Improving the growth performance of Procambarus clarkii includes increasing the final body weight, weight gain rate, growth rate, meat yield, or molting frequency of Procambarus clarkii.

3. Application of ginger extract as a feed additive in improving the antioxidant capacity of Procambarus clarkii.

4. Application of ginger extract as a feed additive in improving the immune defense of red swamp crayfish.

5. Application of ginger extract as a feed additive in improving the air exposure tolerance of Procambarus clarkii.

6. Application of ginger extract as a feed additive in reducing tissue damage and apoptosis induced by air exposure in red swamp crayfish.

7. The application according to any one of claims 1-6, characterized in that, The ginger extract is added at 0.05%-0.80% of the feed weight; The ginger extract is a 70% vol ethanol extract of ginger.

8. A feed for the red swamp crayfish, characterized in that, Includes ginger extract and basic feed; The ginger extract is added at 0.05%-0.80% of the basic feed weight.

9. The Procambarus clarkii feed according to claim 8, characterized in that, The ginger extract is a 70% vol ethanol extract of ginger.

10. The use of the Procambarus clarkii feed according to claim 8 or 9 in any of the following: a. Improve the growth performance of Procambarus clarkii; b. Enhance the antioxidant capacity of Procambarus clarkii; c. Enhance the immune defense capabilities of the red swamp crayfish; d. Improve the air exposure tolerance of Procambarus clarkii; e. Reduces tissue damage and apoptosis induced by air exposure in Procambarus clarkii.

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