Application of reduced glutathione in fish culture

Abstract

The invention discloses application of reduced glutathione in fish culture, and belongs to the technical field of grass carp culture. According to the invention, GSH is added into the grass carp feed, gill injury caused by hypoxia stress is repaired from molecule, cell and tissue levels in multiple dimensions, the feed has anti-oxidation and anti-apoptosis functions, the gill injury of grass carp under hypoxia stress is effectively relieved, the relieving degree is 52-100%, the ability of resisting hypoxia gill injury is enhanced from animals, and the anti-hypoxia gill injury effect is improved. And an efficient, safe and low-cost solution is provided for solving the problem of aquaculture hypoxia.

Description

Technical Field

[0001] The present invention belongs to the technical field of grass carp farming, and specifically relates to the application of reduced glutathione in fish farming. Background Art

[0002] Water is the environment on which aquatic animals depend for survival. Sufficient dissolved oxygen in the water body is a necessary condition for fish to survive, grow, and perform normal physiological functions. In recent years, due to the expansion of aquaculture scale, high feeding and high-density farming, water pollution caused by human activities, and global warming caused by the greenhouse effect, etc., the dissolved oxygen in the water environment has decreased, resulting in a decline in fish feed intake, feed conversion efficiency, and growth, and even causing a large number of fish deaths, seriously hindering the healthy development of the aquaculture industry.

[0003] The harm of hypoxia stress to fish is mainly due to the damage of hypoxia stress to the tissues and organs in the fish body, thus affecting the normal physiological activities of fish. The gill is the main respiratory organ of fish, and its structure is mainly composed of gill arches, gill rakers, gill filaments, and gill lamellae. Among them, the gill lamellae is the place where fish exchange gases with the water environment. The gill is directly in contact with the water environment, so the hypoxic environment will directly affect the structure and function of the gill, showing phenomena such as curling, swelling of gill filaments, and an increase in apoptotic cells. Therefore, improving the hypoxia resistance of fish and alleviating gill damage have become urgent problems to be solved.

[0004] Currently, the prevention of fish hypoxia stress is mainly achieved by turning on aerators, but there are still the following problems in aquaculture production: (1) In order to save costs, the aerator will not be turned on 24 hours a day, and in recent years, extreme weather has occurred frequently, and the problem of hypoxia in intensive farming is very serious; (2) Under intensive farming conditions, when eutrophication and eutrophic algal blooms occur, the dissolved oxygen in the water body drops suddenly, and the aerator cannot resist fish hypoxia stress; (3) Aquaculture often exists in a state of sub-hypoxia.

[0005] Glutathione (GSH) is a functional active tripeptide composed of glutamic acid, cysteine, and glycine, containing γ-amide bond and sulfhydryl group, existing in two forms: reduced form (GSH) and oxidized form (GSSG). Glutathione reductase (GR) can catalyze the interconversion between the two forms, and under physiological conditions, it is mainly in the form of reduced glutathione. Reduced glutathione plays an important role in scavenging free radicals in the body, detoxification, amino acid transmembrane transport, inflammation, and immune processes. Its molecular formula is C 10 H 17 O 6 SN 3 , with a relative molecular mass of 307.33, a melting point of 189 - 193 °C, an isoelectric point of 5.93, soluble in water, dilute alcohol, and liquid ammonia, insoluble in alcohol, ether, and acetone, and its molecular structural formula is shown as follows:

[0006] Summary of the Invention

[0007] In view of the above-mentioned prior art, the present invention provides the application of reduced glutathione in fish farming to solve the technical problem in the prior art that there is no suitable product to solve the gill injury caused by fish under hypoxic stress.

[0008] In order to achieve the above object, the technical solution adopted by the present invention is to provide the application of reduced glutathione in fish farming, specifically in the preparation of a mixture for repairing fish gill injury.

[0009] On the basis of the above technical solution, the present invention can be further improved as follows.

[0010] Further, the above gill injury is the gill injury caused by fish resisting hypoxic stress.

[0011] Further, the above fish is grass carp.

[0012] Further, the above mixture is a feed or a pharmaceutical composition for repairing fish gill injury.

[0013] Further, the above feed is fish feed.

[0014] Further, the addition amount of reduced glutathione in the fish feed is 400 - 500 mg / kg.

[0015] The beneficial effects of the present invention are as follows:

[0016] By exogenously supplementing GSH, the present invention repairs the gill injury caused by hypoxic stress from multiple dimensions at the molecular, cellular and tissue levels, has both antioxidant and anti-apoptosis functions, effectively alleviates the gill injury of grass carp under hypoxic stress, and the alleviation degree is 52 - 100%. It enhances the ability of the animal itself to resist gill injury under hypoxia, and provides an efficient, safe and low-cost solution for the hypoxia problem in aquaculture. Brief Description of the Drawings

[0017] Figure 1 It is the HE staining map (100×) of the gill tissues of grass carp in each treatment group in Example 1. Among them, N-N is the basal diet-normoxia group, N-H is the basal diet-hypoxia group, GSH-H is the diet of the present invention-hypoxia group, ★ represents the detachment of gill epithelial cells, ▲ represents the necrosis of gill epithelial cells, and ● represents the bending of gill lamellae;

[0018] Figure 2Transmission electron micrographs of gill cells of grass carp in each treatment group in Example 1 (4000×, 10000×). Among them, N-N is the basal diet-normoxia group, N-H is the basal diet-hypoxia group, GSH-H is the diet of the present invention-hypoxia group, N is the nucleus, M is the mitochondrion, ER is the endoplasmic reticulum, ERS is endoplasmic reticulum swelling, and AP is the autophagosome;

[0019] Figure 3 Results of TUNEL staining of gill tissue apoptosis in each treatment group in Example 1. Among them, N-N is the basal diet-normoxia group, N-H is the basal diet-hypoxia group, GSH-H is the diet of the present invention-hypoxia group, blue fluorescence indicates the nucleus, and green fluorescence indicates apoptotic cells. Detailed implementation manners

[0020] The following examples are used to make a detailed description of the specific implementation manners of the present invention.

[0021] Example 1

[0022] This example studies the ability of GSH to enhance the resistance of grass carp to gill injury under hypoxic stress.

[0023] 1. Test materials: The GSH used in the test was provided by Anhui Gut New Life Biotechnology Co., Ltd., with a purity of 98%.

[0024] 2. Test location: The test base of the Aquatic Animal Nutrition Research Laboratory of Sichuan Agricultural University.

[0025] 3. Test grouping and test feeds:

[0026] 1) Test grouping: 720 healthy grass carp (Ctenopharyngodon idella) with an average body weight of 11.08 ± 0.01 g were selected for the test and randomly divided into 3 treatment groups: basal diet-normoxia group (N-N), basal diet-hypoxia group (N-H), and diet of the present invention-hypoxia group (GSH-H). There were 6 replicates in each treatment group, and 40 fish in each replicate.

[0027] 2) Test feeds: The basal diet-normoxia group (N-N) and the basal diet-hypoxia group (N-H) were fed the basal diet (N), and the diet of the present invention-hypoxia group (GSH-H) was fed the diet of the present invention (GSH).

[0028] ① Basal diet (N): The basal diet for grass carp was formulated according to the nutritional standards of grass carp formulated feed (GB / T 36205-2018), and its composition and nutritional levels are shown in Table 1:

[0029] Table 1 Basal diet for grass carp

[0030]

[0031]

[0032] Note: The 5% compound premix provides vitamins and trace elements required for the growth of grass carp.

[0033] ② The diet (GSH) of the present invention: In the diet of the present invention, 450 mg (0.45 g) of GSH is added to every 1 kg of the basal diet (the actually measured content in the diet is 437.85 mg / kg of the diet).

[0034] ③ Feed formulation: When formulating the test feed, each feed raw material is ground until it all passes through a 40-mesh sieve, and the material remaining on the 60-mesh sieve is less than 20%. They are mixed in proportion and stirred evenly to make hard pellets with a diameter of 1.5 mm and a length of 3 mm, which are air-dried at room temperature and then stored in a refrigerator at 4°C for standby.

[0035] 4. Test feeding method:

[0036] The test grass carp are respectively raised in cages (1.4 m × 1.4 m × 1.4 m) for 70 days, and are fed 4 times regularly at 7:30, 11:30, 15:30, and 19:30 every day. And the health status of the grass carp is observed every day. During the test period, the water temperature and pH are 29.2 ± 1.8°C and 7.6 ± 0.2 respectively, and the dissolved oxygen in the water is not less than 6.0 mg / L.

[0037] After the feeding test (70 days) is over, a hypoxia stress test is carried out. The basal diet - normoxia group (N-N) is raised under normoxia (Normal) conditions with a dissolved oxygen ≥ 6 mg / L; the basal diet - hypoxia group (N-H) and the diet of the present invention - hypoxia group (GSH-H) are raised under hypoxia (Hypoxia) conditions with a dissolved oxygen of 1 mg / L. The dissolved oxygen concentration of the water body is monitored with a YSI dissolved oxygen meter, and the test period is 96 h.

[0038] During the hypoxia test, the management conditions are the same as those in the growth test. The activities of the test fish are observed and recorded every day. After the hypoxia test is over, the test fish are slaughtered according to the conventional method, and then the gills are separated, and enzyme activity samples, HE section samples, and transmission electron microscope samples are taken respectively.

[0039] 3. Test results:

[0040] Under hypoxia stress, in the grass carp of the basal diet - hypoxia group (N-H), epithelial cell necrosis and floating and shedding occurred in the gills, while the gills of the grass carp in the diet of the present invention - hypoxia group (GSH-H) were normal, comparable to those of the basal diet - normoxia group (N-N); in the grass carp of the basal diet - hypoxia group (N-H), obvious swelling occurred in the endoplasmic reticulum of the gills, while the symptoms of endoplasmic reticulum swelling in the grass carp of the diet of the present invention - hypoxia group (GSH-H) were alleviated.

[0041] Figure 1HE staining images of the gill tissues of grass carp in each treatment group (★ represents detachment of gill epithelial cells, ▲ represents necrosis of gill epithelial cells, ● represents curvature of gill filaments). As can be seen from the figure, in the basal diet - normoxia group (N - N), slight curvature of gill filaments was observed in the gill tissues, the morphology of gill epithelial cells was normal, the structure was intact, and they were closely attached to the gill filaments. After hypoxia stress, symptoms of severe detachment, necrosis of gill epithelial cells, and curvature of gill filaments could be observed in the gill tissues of grass carp in the basal diet - hypoxia group (N - H). However, the gill tissue damage of grass carp caused by hypoxia stress could be completely alleviated in the diet of the present invention - hypoxia group (GSH - H) supplemented with glutathione.

[0042] Figure 2 Transmission electron microscopy images of gill cells of grass carp in each treatment group (N: nucleus; M: mitochondrion; ER: endoplasmic reticulum; ERS: endoplasmic reticulum swelling; AP: autophagosome). As can be seen from the figure, compared with the basal diet - normoxia group (N - N), the gill tissue structure of grass carp in the basal diet - hypoxia group (N - H) was severely damaged, obvious swelling of the endoplasmic reticulum occurred, and a small number of autophagosomes appeared. In the diet of the present invention - hypoxia group (GSH - H) supplemented with glutathione, the symptom of endoplasmic reticulum swelling was alleviated.

[0043] Hypoxia stress can cause cells to produce a large amount of reactive oxygen species (ROS), trigger oxidative stress in cells, lead to lipid peroxidation and protein peroxidation, thereby generating oxidation products such as malondialdehyde (MDA) and protein carbonyl, causing oxidative damage to cells, and then leading to tissue damage. Table 3 shows the indicators related to oxidative damage of gill tissues of grass carp in each treatment group. From the data in the table, it can be known that hypoxia stress led to a significant increase in the contents of ROS, MDA, and PC in the gill tissues. Compared with the basal diet - normoxia group (N - N), the increase amplitudes of ROS, MDA, and PC in the basal diet - hypoxia group (N - H) were 56%, 65%, and 44% respectively. After adding glutathione in the diet of the present invention - hypoxia group (GSH - H), the increase in reactive oxygen species and protein carbonyl caused by hypoxia could be completely alleviated, and lipid peroxidation caused by hypoxia stress was partially alleviated. This indicates that glutathione can completely alleviate the protein oxidative damage of gill tissues caused by hypoxia stress, and the alleviation degree of lipid peroxidation damage reached 52%.

[0044] Table 3 Effects of GSH on indicators related to oxidative damage of gill tissues of juvenile grass carp under hypoxia stress

[0045]

[0046] Note: Data are expressed as mean ± standard deviation (n = 6), and different lowercase letters with different superscripts in the same row indicate significant differences (P < 0.05).

[0047] Under hypoxia stress, the apoptosis of gills of grass carp in the basal diet - hypoxia group (N - H) increased significantly (2.33 times that of the N - N group), while the gill cell apoptosis of grass carp caused by hypoxia stress could be partially alleviated in the diet of the present invention - hypoxia group (GSH - H), and the alleviation degree was 67%.

[0048] Figure 3 Figure 4 shows the TUNEL staining results of gill tissue apoptosis of grass carp in each treatment group (blue fluorescence represents the cell nucleus, and green fluorescence represents apoptotic cells). Table 4 shows the statistical data of apoptosis rates. As Figure 3 can be seen from the data in and Table 4, compared with the basal diet - normoxia group (N - N), the number of apoptotic cells in the basal diet - hypoxia group (N - H) increased significantly (2.33 times) under hypoxia stress. After adding glutathione, the number of apoptotic cells in the diet of the present invention - hypoxia group (GSH - H) decreased significantly (P < 0.05), and the alleviation rate of gill cell apoptosis caused by hypoxia stress reached 67%.

[0049] Table 4 Relative apoptosis rates of gill cells of grass carp in each treatment group

[0050]

[0051] Note: Data are expressed as mean ± standard deviation (n = 6). Different lowercase superscript letters in the same row indicate significant differences (P < 0.05).

[0052] Generally speaking, GSH can alleviate the damage of grass carp gills at different levels under hypoxia stress, and the alleviation degree is 52 - 100%, with good effects.

[0053] Although the specific implementation manners of the present invention have been described in detail in combination with the embodiments, it should not be construed as a limitation on the protection scope of this patent. Within the scope described in the claims, various modifications and deformations that can be made by those skilled in the art without creative labor still fall within the protection scope of this patent.

Claims

1. The application of reduced glutathione in fish farming is characterized by: Application of reduced glutathione in preparing a mixture for repairing fish gill damage.

2. The use according to claim 1, characterized in that: The gill damage is the gill damage caused by the fish's resistance to hypoxic stress.

3. The use according to claim 1, characterized in that: The fish is grass carp.

4. The use according to any one of claims 1 to 3, characterized in that: The mixture is a feed or a medicine composition for repairing fish gill damage.

5. The use according to claim 4, characterized in that: The feed is fish feed.

6. The use according to claim 5, characterized in that: The added amount of the reduced glutathione in the fish feed is 400-500 mg / kg.

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