Application of citral in alleviating transport or heat stress in sea bass

Abstract

This invention discloses the application of citral in alleviating transportation or heat stress in sea bass, belonging to the field of aquaculture stress control technology. The invention discloses the application of citral in the preparation of agents to alleviate transportation and heat stress in sea bass. It alleviates transportation stress by adding citral to the water used for sea bass transportation and heat stress by adding citral to sea bass feed. This invention provides a safe, efficient, and environmentally friendly application scheme for citral, clarifying its optimal concentration and usage method for alleviating transportation and heat stress in sea bass, reducing stress damage, improving sea bass survival rate and quality, and filling the gap in the application of citral in the field of sea bass stress control.

Description

Technical Field

[0001] This invention belongs to the field of aquaculture stress prevention and control technology, and particularly relates to the application of citral in alleviating transportation or heat stress in sea bass. Background Technology

[0002] Lates calcarifer, also known as golden-eyed bass or blind goby, belongs to the Lates genus of the family Latesidae in the order Perciformes. It is a highly valued, euryhaline marine aquaculture fish widely distributed from the Ryukyu Islands to the Indian Ocean. It is favored by fish farmers and consumers for its rapid growth, strong adaptability, delicious flesh, and high nutritional value. In recent years, with the rapid development of intensive and high-density aquaculture, various environmental stresses during the farming process have become increasingly prominent. These include water quality deterioration and frequent disease outbreaks under high-density farming, as well as the stresses of long-distance transportation of fry and marketable fish due to high density and low oxygen levels, and heat stress caused by high summer temperatures. All of these factors lead to severe physiological stress responses in fish, causing oxidative damage, intestinal barrier disruption, and decreased immunity, resulting in reduced survival rates, stunted growth, and decreased quality. This has become a key bottleneck restricting the healthy and sustainable development of the bass aquaculture industry.

[0003] Existing technologies for managing stress in juvenile fish have significant limitations: During transportation, many rely on adding antibiotics to inhibit the growth of aquatic microorganisms, which can easily lead to drug-resistant strains and drug residues in the water, failing to meet the requirements of green aquaculture. Alternatively, chemical anesthetics such as MS-222 are commonly used to reduce the metabolic rate of juvenile fish. While this can temporarily sedate the fish, it presents problems such as high cost, difficulty in controlling the depth of anesthesia, slow recovery, and potential food safety risks. Furthermore, the use of some anesthetics in aquaculture remains controversial regarding compliance. Low-temperature transportation can reduce the metabolic rate of juvenile fish, but the equipment is expensive, and sudden temperature changes can exacerbate stress responses. In the heat stress stage after stocking, conventional measures such as building shade structures and adding groundwater can only locally alleviate the increase in water temperature and cannot fundamentally enhance the juvenile fish's own heat stress resistance.

[0004] Citral is a natural monoterpenoid compound mainly derived from plants such as Litsea cubeba and lemongrass. It has physiological activities such as antibacterial, antioxidant, and anti-inflammatory properties and has been used in livestock, poultry, and some aquatic animals for disease control and growth promotion. However, its application and mechanism of action in alleviating transportation stress and heat stress in sea bass have not been clarified, and no standardized application plan has been formed. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention proposes the application of citral in alleviating transport or heat stress in sea bass.

[0006] To achieve the above objectives, the present invention provides the following technical solution: This invention provides the application of citral in the preparation of a formulation to alleviate transport stress in sea bass.

[0007] Furthermore, the concentration of citral used is 5-20 mg / L, based on the volume of water being transported.

[0008] Furthermore, the concentration of citral used is 5-10 mg / L based on the volume of water being transported.

[0009] This invention also provides the use of citral in the preparation of heat stress relief agents for sea bass.

[0010] Furthermore, the temperature condition for heat stress in the perch is 35°C.

[0011] Furthermore, the dosage of citral used is 0.5 wt% based on feed weight.

[0012] The present invention also provides a formulation for alleviating transport stress and / or heat stress in sea bass, the formulation comprising citral and excipients acceptable for aquaculture.

[0013] The present invention also provides a method for alleviating transport stress in sea bass by adding 5-20 mg / L of citral to the water in which the sea bass are transported.

[0014] The present invention also provides a method for alleviating heat stress in sea bass by adding citral to the sea bass farm feed to alleviate heat stress in sea bass.

[0015] The present invention also provides the application of citral as an anti-stress agent in sea bass farming, wherein the stress includes transportation stress and heat stress.

[0016] Compared with the prior art, the present invention has the following advantages and technical effects: (1) Relieve stress damage: Citral can significantly reduce the antioxidant capacity (SOD, T-AOC) of sea bass under transportation and heat stress, reduce MDA content, and improve the physiological state of the body; (2) Safety and environmental protection: Citral is a natural source (or a synthetic product that meets safety standards), with no drug residues, does not pollute water bodies, and is non-toxic to sea bass, meeting the needs of green aquaculture. (3) Easy to operate: It can be used by adding it to water or feed. The concentration is easy to control, the cost is low, and it is easy to promote and apply on a large scale.

[0017] This invention provides a safe, efficient, and environmentally friendly application solution for citral, clarifying the optimal concentration and application method for alleviating transport stress and heat stress in sea bass, reducing stress damage, improving sea bass survival rate and quality, and filling the application gap of citral in the field of sea bass stress prevention and control. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 The effects of different concentrations of citral on malondialdehyde (MDA) in the gill tissue of sea bass after transport stress and on superoxide dismutase (SOD) after 8 hours of transport. Figure 2 The effect of different concentrations of citral on gill tissue morphology in sea bass after transport stress (HE staining). Figure 3 The effect of different concentrations of citral on the total antioxidant capacity (T-AOC) of liver tissue in sea bass after heat stress; Figure 4 The effect of different concentrations of citral on the gill tissue morphology of sea bass after heat stress (HE staining). Detailed Implementation

[0020] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0021] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the present invention will be further described in detail below with reference to specific embodiments.

[0022] Unless otherwise specified, the raw materials used in the embodiments of this invention were all purchased through commercial channels; unless otherwise specified, all groups in the embodiments were managed under conventional breeding and feeding standards.

[0023] 1. Experimental Materials and Instruments Materials: Healthy sea bass (size: weight 1-2g, body length 4-6cm), citral (purity ≥95%), aquaculture water (meeting fishery water standards); among which, citral is selected from natural extraction or artificial synthesis, with a purity ≥95%, and is a colorless to pale yellow oily liquid, odorless and free of impurities, meeting the safety standards for aquatic feed or water additives; the citral used in this invention was purchased from Sigma-Aldrich, product number C80037; Instruments: dissolved oxygen meter, thermometer, centrifuge, enzyme-linked immunosorbent assay (ELISA) reader, dissecting instruments, etc.

[0024] 2. Application of citral in alleviating transport stress in sea bass Adding during transportation: Mix citral with the transport water to maintain a concentration of 5-10 mg / L, and control the temperature at 20-26℃. This method is suitable for transporting live animals (37 animals / m³) and can last for 8-24 hours.

[0025] 3. Application of citral in alleviating heat stress in sea bass When the temperature of the aquaculture water exceeds the critical temperature for heat stress, adding 0.5 wt% citral to the feed can effectively alleviate the stress response caused by high temperature.

[0026] Example 1: An experiment on the relief of transport stress in sea bass by citral Groups: control group (transport water without citral), experimental group 1 (with 5 mg / L citral), experimental group 2 (with 10 mg / L citral), experimental group 3 (with 20 mg / L citral), and a blank control group (aquaculture water without citral and without transportation stress treatment). Each group had 3 replicates, with 37 bass per replicate. Treatment: All bass in the control group and experimental groups 1-3 were temporarily held in water with the corresponding concentration of citral for 1 hour before being transported at high density (37 fish / m³) at a temperature of 20℃. The blank control group was not fed citral and was sampled together with the other transport stress treatment groups after the completion of transport. The survival rate of bass in each group was measured at 4h, 8h, 12h, and 24h of transport time, and samples were taken from each group for the following indicators (7 fish were randomly selected from each replicate, for a total of 21 fish per group).

[0027] Detection indicators: After transportation, the survival rate, SOD and MDA content of each group of bass were measured; and the effect of different concentrations of citral on the gill tissue morphology of bass after transportation stress was detected (HE staining). Specific procedures are as follows: Gill tissue samples from each group of bass were placed in 0.9% physiological saline (1:9 dilution) and homogenized using a tissue homogenizer. The homogenate was centrifuged at 3000×g for 10 minutes at 4℃, and the supernatant was collected for later use. Antioxidant enzyme activities, namely superoxide dismutase (SOD) and malondialdehyde (MDA) content, were tested. All enzyme activities were performed using a commercially available kit (provided by Nanjing Jiancheng Bioengineering Institute), and the specific methods were described in the manufacturer's instructions.

[0028] Each group of fish gill samples was embedded and sectioned in paraffin. The specific procedures included rinsing with running water, dehydration with gradient alcohol, clearing with xylene, paraffin embedding, 4μm sectioning, staining with hematoxylin and eosin, and observation and image acquisition under an optical microscope.

[0029] The test results are shown in Table 1 and Figures 1-2As shown, the control group, which did not receive citral, had a 100% survival rate. However, based on subsequent antioxidant indicators and tissue sections, it was determined that the experimental fish experienced sublethal damage after simulated transportation. Adding an appropriate amount of citral essential oil (5-10 mg / L) improved their condition. When 20 mg / L citral essential oil is added directly to the transport water, there is a clear safety threshold: low concentrations (5-10 mg / L) exhibit a stress-protective effect, while high concentrations (20 mg / L) exceed the tolerance range of the bass, causing acute toxicity and leading to a significant decrease in survival rate during transportation. Therefore, a citral concentration of 20 mg / L is not suitable for the water transportation of bass.

[0030] Table 1. Effects of different concentrations of citral on the survival rate of sea bass after transport stress. Note: "—" indicates that all the experimental fish in this group have died.

[0031] Example 2: An experiment on the relief of heat stress in sea bass by citral Groups were established: an alcohol control group (no citral added, no heat stress treatment), a citral control group (0.5% citral added, no heat stress treatment), an alcohol experimental group (no citral added, but heat stress treatment), and a citral experimental group (0.5% citral added, and heat stress treatment). Each group had three replicates, with 37 sea bass per replicate. In both the alcohol control and alcohol experimental groups, the alcohol added was the same amount of citral as the solvent.

[0032] Treatment: The bass in each group were kept in normal water at 25℃. The feed-added group was fed citral-containing feed for 7 days. Then, the bass in each experimental group were placed in high-temperature water at 35℃ for 24 hours for heat stress treatment.

[0033] After heat stress, the survival rate of bass in each group was tested, and samples were taken from each group to perform the following tests (7 fish were randomly selected from each replicate, for a total of 21 fish per group). The specific procedures are as follows: The liver tissue samples from each group of sea bass were placed in 0.9% physiological saline (1:9 dilution) and homogenized using a tissue homogenizer. The homogenate was centrifuged at 3000×g for 10 minutes at 4℃, and the supernatant was collected for later use. The total antioxidant capacity (T-AOC) in the tissue was tested using a commercial kit (provided by Nanjing Jiancheng Biotechnology Institute), and the specific method was as described in the instruction manual.

[0034] The gill tissues of the bass in each group were embedded and sectioned in paraffin. The specific procedures included rinsing with running water, dehydration with graded alcohol, clearing with xylene, paraffin embedding, sectioning into 4μm sections, staining with hematoxylin and eosin, and observation and image acquisition under an optical microscope.

[0035] The results are shown in Table 2 and Figures 3-4 As shown, the survival rate of the citral group was higher than that of the alcohol group, indicating that adding citral can reduce the effects of heat stress. Furthermore, the addition of citral significantly reduced the degree of oxidative damage in sea bass and improved their heat stress status.

[0036] Table 2. Effects of different concentrations of citral on the survival rate of sea bass after heat stress. The above are merely preferred embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. Application of citral in the preparation of a formulation to alleviate transport stress in sea bass.

2. The application according to claim 1, characterized in that, The concentration of citral used is 5-20 mg / L, based on the volume of water transported.

3. The application according to claim 2, characterized in that, The concentration of citral used is 5-10 mg / L, based on the volume of water transported.

4. Application of citral in the preparation of heat stress relief agents for sea bass.

5. The application according to claim 4, characterized in that, The temperature condition for heat stress in the perch was 35°C.

6. The application according to claim 4, characterized in that, The dosage of citral used is 0.5 wt% based on feed weight.

7. A formulation for alleviating transport stress and / or heat stress in sea bass, characterized in that, The formulation contains citral and other aquaculture-acceptable excipients.

8. A method for alleviating transport stress in sea bass, characterized in that, Add 5-20 mg / L of citral to the water used for transporting bass.

9. A method for alleviating heat stress in sea bass, characterized in that, Adding citral to sea bass feed can alleviate heat stress in sea bass.

10. The application of citral as an anti-stress agent in sea bass aquaculture production, characterized in that, The stresses include transportation stress and heat stress.

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