Application of linoleic acid in preparation of feed for relieving salinity stress of fishes

Abstract

The invention provides an application of linoleic acid in preparation of a feed for relieving fish salinity stress. According to the application of the linoleic acid in preparation of the feed for relieving the salinity stress of the fishes, related experiments verify that the addition of the linoleic acid enhances the overall tolerance of the fishes to the salinity stress, and under the stress environment that the salinity continuously rises, the stress resistance of the fishes to the salinity stress is improved. Fish has better growth performance, lower physiological stress level, stronger antioxidant ability and immune response and healthier tissue structure, and is beneficial to increase of culture benefits of freshwater fish and marine fish.

Description

Technical Field

[0001] The present invention relates to the technical field of aquaculture, and particularly to the application of linoleic acid in the preparation of feed for alleviating salinity stress in fish. Background Art

[0002] The aquaculture of freshwater fish and seawater fish faces many challenges during the rapid development process. Among them, salinity stress is a key issue affecting the fish farming efficiency. When fish encounter sudden changes in environmental salinity, such as the conversion between fresh water and seawater, or salinity fluctuations caused by heavy rain, it will trigger serious physiological imbalances: the disorder of water and ion exchange in the fish body leads to osmotic pressure disorder, the abnormal ion transport function of gill tissues causes electrolyte metabolism imbalance, and at the same time, the impaired immune system function makes the disease resistance of fish significantly decline. These chain reactions directly lead to slow fish growth, reduced feed utilization rate and increased mortality, causing significant economic losses.

[0003] Currently, the traditional mitigation methods that rely on artificial gradient salinity adjustment or electrolyte addition are not only cumbersome to operate and difficult to scale up, but also have the risks of unstable effects and side effects caused by long-term use. Therefore, there is an urgent need to develop a new method for safely and efficiently regulating salinity stress through the feed approach. Summary of the Invention

[0004] The present invention provides the application of linoleic acid in the preparation of feed for alleviating salinity stress in fish. Through relevant experimental verification, the addition of linoleic acid enhances the overall tolerance of fish to salinity stress. Under the stress environment of continuously increasing salinity, fish have better growth performance, lower physiological stress levels, stronger antioxidant capacity and immune responses, as well as healthier tissue structures, which is beneficial to increasing the aquaculture benefits of freshwater fish and seawater fish.

[0005] According to one aspect of the present invention, there is provided the application of linoleic acid in the preparation of feed for alleviating salinity stress in fish.

[0006] Fish salinity stress refers to the phenomenon of systemic physiological function collapse in cultured fish when the water salinity changes suddenly in a short period and exceeds its own regulation ability. Its hazards are manifested as fish feeding stagnation, metabolic disorders, and a cliff-like decline in immunity, ultimately leading to growth stagnation and batch death, causing significant economic losses to farmers. As described in the background art, currently, the traditional mitigation methods that rely on artificial gradient salinity adjustment or electrolyte addition are not only cumbersome to operate and difficult to scale up, but also have the risks of unstable effects and side effects caused by long-term use. Therefore, developing a new method for safely and efficiently regulating salinity stress through the feed approach is of great significance for the healthy ecological development of the freshwater fish and seawater fish aquaculture industries.

[0007] Linoleic acid is a ω-6 polyunsaturated fatty acid naturally present in vegetable oils such as corn oil and soybean oil. For a long time, it has only been used for basic nutritional fortification in the field of animal breeding, improving breeding efficiency by regulating lipid metabolism, enhancing immune function, and promoting growth and development. In recent years, linoleic acid has been applied in alleviating heat stress in livestock and poultry. However, there are few reports on its specific mechanism of action and actual application effect in the regulation of salinity stress in freshwater fish and marine fish.

[0008] The present invention uses linoleic acid to prepare a feed for alleviating salinity stress in fish. Through relevant experimental verification, the addition of linoleic acid enhances the overall tolerance of fish to salinity stress. In a stress environment with continuously increasing salinity, fish have better growth performance, lower physiological stress levels, stronger antioxidant capacity and immune response, and healthier tissue structure, which is beneficial to increasing the breeding efficiency of freshwater fish and marine fish.

[0009] Preferably, the above-mentioned fish include freshwater fish.

[0010] Preferably, the above-mentioned freshwater fish include grass carp.

[0011] Preferably, the above-mentioned fish include marine fish.

[0012] Preferably, the above-mentioned marine fish include turbot.

[0013] Preferably, calculated by mass percentage, the feed includes 0.1 - 1.0% linoleic acid.

[0014] Preferably, calculated by mass percentage, the feed includes 0.5% linoleic acid.

[0015] Preferably, calculated by mass percentage, the feed further includes 30 - 50% protein feed, 20 - 40% carbohydrate feed, 1 - 5% vitamin and mineral feed, and 1 - 30% basic feed.

[0016] Preferably, the protein feed includes at least one of wheat gluten, soybean meal, casein, and gelatin.

[0017] Preferably, the carbohydrate feed includes flour.

[0018] Preferably, the basic feed includes at least one of corn, wheat, wheat bran, and grass powder.

[0019] Preferably, the application includes at least one of the following a, b, c, d, and e: a. The salinity stress of fish is to improve the growth performance of fish under salinity stress; b. The salinity stress of fish is to improve the physiological indexes of fish under salinity stress; c. The salinity stress of fish is to improve the immune function of fish under salinity stress; d. The salinity stress of fish is to improve the antioxidant effect of fish under salinity stress; e. The salinity stress of fish is to reduce the degree of tissue damage of fish under salinity stress.

[0020] Preferably, a. the salinity stress on fish is to increase the specific growth rate, feed conversion rate, and average daily feed intake of fish under salinity stress; b. the salinity stress on fish is to reduce the levels of glucose and lactic acid in the blood and plasma osmotic pressure of fish under salinity stress; c. the salinity stress on fish is to increase the serum immunoglobulin level and lysozyme activity of fish under salinity stress; d. the salinity stress on fish is to reduce the content of malondialdehyde in the liver tissue of fish under salinity stress and increase the activities of superoxide dismutase, glutathione peroxidase, and catalase in the liver tissue of fish; e. the salinity stress on fish is to reduce the degree of damage to the gill tissue and liver cells of fish under salinity stress. Description of the Drawings

[0021] Figure 1 Shows the effects of different concentrations of LA (linoleic acid) in Example 1 on the viability of two cell lines (PSF and CIK); Figure 2 Shows the effects of different treatment conditions in Example 2 on the cell viability of two cell lines (PSF and CIK); Figure 3 Shows the lactate dehydrogenase (LDH) activities of two cell lines (PSF and CIK) under different treatment conditions in Example 3. Detailed Description of the Invention

[0022] The technical features in the technical solutions provided by the present invention will be further clearly and completely described below in conjunction with the detailed description of the invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative efforts shall fall within the protection scope of the present invention.

[0023] The quantitative experimental data involved in the following examples are expressed as means.

[0024] Analysis of variance (ANOVA): used to compare the differences between the experimental group and the control group.

[0025] Multiple comparisons: Tukey HSD test was used for multiple comparisons to determine significant differences.

[0026] Correlation analysis: analyze the correlation between growth performance indicators and physiological indicators.

[0027] Linoleic acid (CAS No.: 60-33-3), the source of linoleic acid used in this solution is soybean oil, and about 50 g to 60 g of linoleic acid (i.e., 50% to 60%) is contained in 100 g of soybean oil.

[0028] Example 1 Drug Toxicity Experiment of Linoleic Acid 1. Main Instruments and Reagents Microplate reader, PBS buffer, 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt solution (CCK-8 solution); Grass carp ( Ctenopharyngodon Idella ) snout fibroblast cells (Grass Carp Snout Fibroblast cells, PSF) are stored in this laboratory. The public can obtain them from the applicant and are only used for repeating the experiments of this invention. Grass carp ( Ctenopharyngodon Idella ) kidney cells (Grass Carp Kidney cells, CIK) are stored in this laboratory. The public can obtain them from the applicant and are only used for repeating the experiments of this invention.

[0029] 2. Experimental methods PSF and CIK cells are respectively inoculated into 96-well culture plates at a density of 1×10 5 cells / well and cultured at 28°C for 24 hours. Linoleic acid is diluted with medium into different concentrations (200 μM, 100 μM, 50 μM, 25 μM, 0 μM). Different concentrations of linoleic acid are added to the 96-well plates and co-incubated with PSF and CIK cells at 28°C. After 24 hours, the medium containing linoleic acid is discarded, and then the cells in each well are washed with PBS buffer. Subsequently, 100 μL of serum-free medium solution containing 1:10 CCK-8 is added and incubated in the dark at room temperature for 2 hours. The wavelength of the microplate reader is set to 450 nm, and the absorbance of each well is measured and the cell viability is calculated.

[0030] The calculation formula of cell viability is as follows: .

[0031] 3. Experimental results The results of cell viability are as Figure 1 shown. In the range of linoleic acid concentration from 0 to 200 μM, there is no significant difference in cell viability between the linoleic acid treatment group and the control group (P>0.05), indicating that linoleic acid has no toxic effect on grass carp PSF and CIK cells in this concentration range.

[0032] Example 2 Protective effect of linoleic acid on cell viability under salinity stress 1. Main instruments and reagents Dimethyl sulfoxide (DMSO). The other instruments and reagents used in this example are the same as those in Example 1.

[0033] 2. Experimental methods PSF and CIK cells are respectively inoculated into 96-well culture plates at a density of 1×10 5Cells were inoculated into a 96-well culture plate at a density of [number of cells / holes] and cultured at 28 °C for 24 hours. In the linoleic acid treatment group, linoleic acid was diluted to 100 μM with culture medium and then added to the 96-well plate. It was co-incubated with PSF and CIK cells at 28 °C for 4 hours, and then a sodium chloride solution with a concentration of 5 mM was added. The salinity of the system was 80 μM, and the total volume of the system was 100 μL. In the control group, no linoleic acid was added, the salinity of the system was 0, and the total volume of the system was 100 μL. In the salinity stress group, no linoleic acid was added, the salinity of the system was 80 μM, and the total volume of the system was 100 μL. In the vehicle control group, DMSO with the same volume as linoleic acid was added, the salinity of the system was 80 μM, and the total volume of the system was 100 μL. In the linoleic acid treatment group, the final concentration of linoleic acid was 80 μM, and the total volume of the system was 100 μL. After 24 hours, in the control group (Control), salinity stress group (SaltStress), vehicle control group (Vehical), and linoleic acid treatment group (LA), the saline culture medium was discarded, and the cells in each well were washed with PBS buffer. Subsequently, 100 μL of serum-free medium solution containing 1 / 10 CCK8, a 1:10 CCK-8 serum-free medium solution, was added, and it was incubated in the dark at room temperature for 2 hours. The wavelength of the microplate reader was set to 450 nm, and the absorbance of each well was measured and the cell viability was calculated.

[0034] The calculation formula for cell viability is as follows: 。

[0035] 3. Experimental results The results of cell viability are as Figure 2 shown. The cell viability in the salinity stress group decreased significantly (P < 0.05), while the cell viability in the linoleic acid treatment group was significantly higher than that in the salinity stress group (P < 0.05), indicating that linoleic acid can effectively alleviate the decrease in cell viability caused by salinity stress.

[0036] Example 3 Repair effect of linoleic acid on cell membrane damage induced by salinity stress 1. Main instruments and reagents Lactate dehydrogenase (LDH) detection kit. The other instruments and reagents used in this example are the same as those in Example 2.

[0037] 2. Experimental method PSF and CIK cells were respectively seeded at a density of 1×10 5Cells were inoculated into a 96-well culture plate at a density of [number of cells / well], and cultured at 28 °C for 24 hours. In the linoleic acid treatment group, linoleic acid was diluted to 100 μM with culture medium and then added to the 96-well plate. It was co-incubated with PSF and CIK cells at 28 °C for 4 hours, and then a sodium chloride solution with a concentration of 5 mM was added. The salinity of the system was 80 μM, and the total volume of the system was 100 μL. In the positive control group, linoleic acid was not added, the salinity of the system was 0, and the total volume of the system was 100 μL. In the control group, linoleic acid was not added, the salinity of the system was 0, and the total volume of the system was 100 μL. In the salinity stress group, linoleic acid was not added, the salinity of the system was 80 μM, and the total volume of the system was 100 μL. In the vehicle control group, DMSO with the same volume as linoleic acid was added, the salinity of the system was 80 μM, and the total volume of the system was 100 μL. In the linoleic acid treatment group, the final concentration of linoleic acid was 80 μM, and the total volume of the system was 100 μL. After 22 hours in the positive control group (Positive), control group (Control), salinity stress group (SaltStress), vehicle control group (Vehical), and linoleic acid treatment group (LA), 10 μL of LDH release reagent was added to the positive control group. After 2 hours, the culture media in the culture wells of the five groups were collected by centrifugation respectively (another culture medium with the same volume that had not been cultured with cells was used as the background blank control group for subsequent LDH activity calculation). Subsequently, LDH detection reagent solution was added, and it was incubated in the dark at room temperature for 30 minutes, and the absorbance at a wavelength of 490 nm was measured. The detection process was carried out according to the instructions of the LDH kit to determine the activity of lactate dehydrogenase (LDH). Calculate the LDH activity (%) = ((OD value of the experimental group - OD value of the background blank control group) / (OD value of the positive control group - OD value of the background blank control group)) × 100%.

[0038] 3. Experimental Results The results of LDH activity are as Figure 3 shown. Compared with the positive control group, the LDH activity in the linoleic acid treatment group decreased significantly, indicating that linoleic acid can effectively protect the integrity of the cell membrane and reduce the damage caused by salinity stress.

[0039] Example 4: Aquaculture Experiment on the Alleviation of Salinity Stress in Grass Carp by Linoleic Acid-Containing Feed 1. Main Instruments and Reagents Aquaculture tanks: with a volume of 1000 L, 1 independent aquaculture tank was used for each group's repeated experiment, salinity meter, water quality detector, blood collection equipment, growth measurement tools; Grass carp ( Ctenopharyngodon Idella ) were obtained commercially. The grass carp used were in good health and weighed about 100 - 150 g; Common feed: Commercially available special feed for grass carp, the nutritional components meet the growth requirements of grass carp, and this common feed does not contain linoleic acid; Linoleic acid feed: Calculated by mass percentage, the linoleic acid feed includes 39.1% protein feed (composed of 3.91% gluten, 19.55% soybean meal, 7.82% casein, and 7.82% gelatin), 30% carbohydrate feed (the above carbohydrate feed is flour), 3% vitamin and mineral feed (composed of 0.006% vitamin A, 0.006% vitamin D, 0.003% vitamin E, 0.006% vitamin C, 0.0045% vitamin B complex, 0.45% calcium, 0.21% phosphorus, 0.06% magnesium, 0.006% zinc, 0.006% iron, 0.003% copper, 0.006% manganese, 0.0006% iodine, 0.00006% selenium, and the balance carrier), 0.5% linoleic acid, and 27.4% basic feed (composed of 13.7% corn, 8.22% wheat, 2.74% wheat bran, and 2.74% grass meal).

[0040] Malondialdehyde (MDA) was purchased from MyBioSource, superoxide dismutase (SOD), and glutathione peroxidase (GPx) were purchased from Cayman Chemical, and the catalase (CAT) detection kit, glucose, lactic acid, and osmotic pressure kits were purchased from Sigma - Aldrich.

[0041] 2. Experimental methods (1) This example was divided into an experimental group and a control group. The above - mentioned linoleic acid feed was used in the experimental group, and the above - mentioned ordinary feed was used in the control group. Three replicate experiments were set up for both the experimental group and the control group. 30 grass carps were put in each replicate experiment. A total of 180 grass carps were used in the experimental group and the control group.

[0042] (2) Salinity stress procedure The total experimental period was 31 days, starting from the time when grass carps were put into the aquaculture tank. In chronological order, it was divided into an adaptation period (with a duration of 7 days and a salinity of 0 ppt), a salinity change period (with a duration of 10 days, the initial salinity of the salinity change period was 1 ppt, the salinity increase rate was 1 ppt / day, and the target salinity was 10 ppt), and a maintenance period (with a duration of 14 days and a salinity of 10 ppt).

[0043] (3) Feeding management Throughout the experimental period, feeding was carried out 2 times a day, at 8:00 am and 6:00 pm respectively. The feeding amount was adjusted according to the weight and feeding situation of the grass carps, and the feeding amount was 3% - 5% of the weight of the grass carps at the time of feeding. The feeding method was to evenly sprinkle the feed on the surface of the aquaculture tank to ensure that all grass carps could feed.

[0044] (4) Water quality management During the entire experimental period, the water temperature in the aquaculture tanks was maintained at 25 - 28 °C, the dissolved oxygen content was > 5 mg / L, the ammonia nitrogen concentration was < 0.5 mg / L, the water change frequency was once a week, and the water change volume each time was 30% of the tank volume.

[0045] (5)Sampling points The sampling points included Day 0 (before the experiment), Day 10 (end of the salinity change period), Day 24 (end of the maintenance period), and Day 31 (end of the experiment).

[0046] (6)Detection methods Body weight: Weigh the fish in each group at the sampling points and calculate the average fasting body weight of the fish in each group.

[0047] Specific growth rate (SGR) / % = (ln final weight - ln initial weight) / culture time × 100.

[0048] Feed conversion rate (FCR) / % = feed consumption / fish weight gain × 100.

[0049] The detection experiments of glucose level, lactic acid level, osmotic pressure, MDA content, SOD activity, GPx activity, CAT activity, immunoglobulin level, and lysozyme activity were carried out according to the steps in the instructions of the corresponding kits.

[0050] Degree of gill tissue and liver damage: At the end of the experiment, take samples of the middle complete gill filaments of the gill arches and the left lobe of the liver of grass carp in the control group and the experimental group (size about 5 × 5 × 3 mm), and immediately immerse them in Bouin's fixative (for gill tissue) and 10% neutral buffered formalin (for liver) for 24 hours; after dehydration with gradient ethanol and paraffin embedding, prepare continuous sections with a thickness of 5 μm, and stain them using hematoxylin - eosin (H&E) staining method. Use an optical microscope (400×) to conduct histological observation and damage degree grading on 5 randomly selected fields of view: the gill tissue is comprehensively evaluated based on the degree of secondary gill lamella edema, the ratio of epithelial cell separation / necrosis, capillary congestion status, and the morphological changes of chloride cells; the liver is judged according to the area of hepatocyte fatty vacuolization, the number of karyopyknosis / karyolysis, the integrity of hepatic cord structure, and the range of inflammatory cell infiltration. The damage grades are divided into four levels: no damage (normal tissue structure, pathological features ≤ 5%), mild (local pathological changes, affected range 5% - 30%), moderate (multifocal lesions, affected range 30% - 60%), and severe (diffuse damage, affected range > 60%).

[0051] 3. Experimental results Table 1 Growth performance test of the test subjects

[0052] Table 2 Physiological index test of the test subjects

[0053] Table 3 Antioxidant effect test of the test subjects

[0054] Table 4 Immune function test of the test subjects

[0055] Table 5 Tissue damage degree test of the test subjects

[0056] Under continuous salinity stress, the comprehensive tolerance of grass carp in the linoleic acid addition group (experimental group) was better than that of the control group. The experimental group maintained stable feeding activity and growth rate, and its specific growth rate was significantly higher than that of the control group (P<0.05), and the feed conversion efficiency was also significantly better than that of the control group (P<0.05); at the same time, the experimental group effectively alleviated the metabolic disorder caused by osmotic imbalance, and its blood glucose level was significantly lower than that of the control group (P<0.05), the lactic acid level was significantly lower than that of the control group (P<0.05), and the plasma osmotic pressure was significantly lower than that of the control group (P<0.05) and tended to be stable. The experimental group activated the endogenous antioxidant defense system systemically, and its superoxide dismutase activity was significantly higher than that of the control group (P<0.05), glutathione peroxidase activity was significantly higher than that of the control group (P<0.05), catalase activity was significantly higher than that of the control group (P<0.05), and the content of malondialdehyde, an oxidative damage marker, was significantly lower than that of the control group (P<0.05); the immunoglobulin synthesis ability of the experimental group was significantly higher than that of the control group (P<0.05), and the lysozyme activity was also significantly higher than that of the control group (P<0.05). The above physiological regulations were finally reflected in the improvement of the structural integrity of key organs: the gill filament epithelial tissue in the experimental group was arranged tightly (the damage in the control group was significantly more serious, P<0.05), the liver cells had normal fat metabolism (the abnormality in the control group was more obvious, P<0.05), and the overall tissue damage degree was significantly lower than that of the control group (P<0.05). From the above results, it can be seen that the addition of linoleic acid enhanced the overall tolerance of fish to salinity stress. In the stress environment of continuously increasing salinity, fish had better growth performance, lower physiological stress levels, stronger antioxidant capacity and immune response, and healthier tissue structure, which was beneficial to increasing the breeding benefits of freshwater fish and seawater fish.

[0057] The above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. Although the present invention has been described in detail with reference to the above embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced, but these modifications or replacements are all within the protection scope of the present invention.

Claims

1. Use of linoleic acid in preparing feed for alleviating salinity stress in fish.

2. The use of linoleic acid in the preparation of a feed for relieving salinity stress in fish according to claim 1, wherein The fish includes freshwater fish.

3. The use of linoleic acid in the preparation of a feed for relieving salinity stress in fish according to claim 2, wherein The freshwater fish includes grass carp.

4. The use of linoleic acid in the preparation of a feed for relieving salinity stress in fish according to claim 1, characterized in that, The fish includes seawater fish.

5. The use of linoleic acid in the preparation of a feed for alleviating salinity stress in fish as claimed in claim 4, wherein, The seawater fish includes turbot.

6. The use of linoleic acid in the preparation of a feed for relieving salinity stress in fish as claimed in claim 1, wherein Calculated by mass percentage, the feed includes 0.1 - 1.0% linoleic acid.

7. Use of linoleic acid in the preparation of a feed for relieving salinity stress in fish, characterized in that, Calculated by mass percentage, the feed includes 0.5% linoleic acid.

8. The use of linoleic acid in the preparation of a feed for relieving salinity stress in fish according to claim 6, characterized in that, Calculated by mass percentage, the feed further includes 30 - 50% protein feed, 20 - 40% carbohydrate feed, 1 - 5% vitamin and mineral feed, 1 - 30% basic feed.

9. The application according to any one of claims 1 to 8, characterized in that, The application includes at least one of the following a, b, c, d, e: a. The salinity stress in the fish is to improve the growth performance of fish under salinity stress; b. The salinity stress in the fish is to improve the physiological indexes of fish under salinity stress; c. The salinity stress in the fish is to improve the immune function of fish under salinity stress; d. The salinity stress in the fish is to improve the antioxidant effect of fish under salinity stress; e. The salinity stress in the fish is to reduce the degree of tissue damage of fish under salinity stress.

10. The application according to claim 9, wherein The application includes at least one of the following a, b, c, d, e: a. The salinity stress in the fish is to improve the specific growth rate, feed conversion rate, and average daily food intake of fish under salinity stress; b. The salinity stress in the fish is to reduce the levels of glucose, lactic acid in the blood and plasma osmotic pressure of fish under salinity stress; c. The salinity stress in the fish is to improve the serum immunoglobulin level and lysozyme activity of fish under salinity stress; d. The salinity stress in the fish is to reduce the content of malondialdehyde in the liver tissue of fish under salinity stress and improve the activities of superoxide dismutase, glutathione peroxidase, and catalase in the liver tissue of fish; e. The salinity stress in the fish is to reduce the degree of damage to the gill tissue and liver cells of fish under salinity stress.

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