Composition for improving ammonia nitrogen stress resistance of micropterus salmoides as well as preparation method and application of composition
By preparing compositions of turmeric, vine tea and mulberry leaf extract, the ammonia nitrogen stress problem faced by largemouth bass under high-density farming was solved, which significantly improved the anti-ammonia nitrogen ability of fish, reduced mortality and stress response, and enhanced immune function and metabolic balance.
Patent Information
- Application Number
- CN202510367863.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-26
- Publication Date
- 2025-06-20
- Estimated Expiration
- 2045-03-26
AI Technical Summary
Largemouth bass faces the challenge of ammonia nitrogen pollution under the high-density and intensive breeding model, resulting in impeded growth and development, low immune function and tissue damage. It is difficult for the existing technology to effectively prevent and reverse the damage caused by ammonia nitrogen stress.
By preparing a composition, including extracts of turmeric, rattan tea and mulberry leaves, turmeric oil resin and rattan tea extract are prepared by ethanol extraction and crystallization, combined with mulberry leaf extract, a composition with anti-ammonia nitrogen stress effect is formed, and it is added to fish feed.
This composition significantly improves the resistance of largemouth bass to ammonia nitrogen stress, reduces the mortality rate of fish, maintains the activity of gill filaments Na+/K+-ATPase, alleviates the stress response, reduces the damage to the liver by ammonia nitrogen stress, and enhances the immune function and metabolic balance of fish.
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Figure CN120168595A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the field of biotechnology, and in particular to a composition for improving the ability of largemouth bass to resist ammonia nitrogen stress, a preparation method and application thereof. Background Art
[0002] In the aquaculture industry, ammonia nitrogen is considered one of the key environmental factors affecting the health and growth of aquatic animals, especially in high-density intensive farming models. In this model, the accumulation of aquatic animal feces, leftover feed, and excessive fertilizer application are all rich in protein. These proteins are converted into ammonia nitrogen by microbial decomposition in the water, resulting in a significant increase in the concentration of ammonia nitrogen in the water. Ammonia nitrogen enters the fish body mainly in the form of molecular ammonia (NH3, non-ionic ammonia) through the fish gills, epidermis, intestinal mucosa and other tissues, posing a potential toxic threat to fish.
[0003] The negative impacts of ammonia nitrogen poisoning on farmed fish are extensive and far-reaching, including but not limited to slowed growth, weakened immune system function, increased oxidative stress response, and frequent inflammatory responses. These physiological disorders not only inhibit the normal growth and development of fish, but may also cause metabolic disorders, tissue damage, and even death. The concentration of ammonia nitrogen in the aquaculture water may fluctuate rapidly, posing an acute stress to fish, leading to hyperplasia of gill filament tissue and thickening of the mucus cell layer, which in turn weakens the blood's oxygen-carrying capacity and liver function, manifesting as symptoms such as tissue edema.
[0004] The high-density intensive farming model of largemouth bass also faces the challenge of ammonia nitrogen pollution. This farming method has exacerbated the sharp increase in ammonia nitrogen concentration in the water, posing a serious threat to the growth and development of largemouth bass. Studies have shown that ammonia nitrogen stress not only interferes with the ammonia excretion mechanism of largemouth bass and reduces its decomposition and metabolic activities, but also damages important tissue structures such as gills and liver, increases serum transaminase activity, and inhibits its immune and antioxidant capacity.
[0005] At present, most of the countermeasures for excessive ammonia nitrogen concentration in aquaculture water are initiated only after fish die or excessive ammonia nitrogen is detected, using water quality regulators such as probiotics, organic acids or adsorbents for intervention. However, such delayed treatment strategies are often difficult to reverse the damage caused to fish by ammonia nitrogen stress, and the resistance of largemouth bass is reduced, making them more susceptible to pathogenic microorganisms. Therefore, the development of a composition that can effectively enhance the ability of largemouth bass to resist ammonia nitrogen stress is of great significance for preventing ammonia nitrogen poisoning, ensuring the healthy growth of fish, and reducing aquaculture losses. Summary of the invention
[0006] In view of this, the present invention proposes a composition for improving the ability of largemouth bass to resist ammonia nitrogen stress, a preparation method and application thereof.
[0007] The technical solution of the present invention is realized as follows:
[0008] In a first aspect, the present invention provides a method for preparing a composition for improving the ammonia nitrogen stress resistance ability of Micropterus salmoides, comprising the following steps:
[0009] (S1) Add 8-fold volume of 85% v / v ethanol solution to 28-32 parts by weight of turmeric, reflux extract 2-4 times, each time for 1.5-3 h, collect the extract and concentrate it to obtain a thick paste; add 1-fold volume of 70% v / v ethanol solution to the thick paste, crystallize at 4°C, stand for 12-36 h, collect the supernatant, and concentrate it to a relative density of 1.10-1.15 to obtain turmeric oleoresin;
[0010] (S2) Add 8-fold volume of 85% v / v ethanol solution to 28-32 parts by weight of Ampelopsis grossedentata, reflux extract 2-4 times, each time for 1.5-3 h, and combine the extracts of each time to obtain an Ampelopsis grossedentata extract;
[0011] (S3) Add 10-fold volume of deionized water to 8-12 parts by weight of mulberry leaves, place in a boiling water bath, extract 2-4 times, each time for 1.5-3 h, and combine the extracts of each time to obtain a mulberry leaf extract;
[0012] (S4) Mix the Ampelopsis grossedentata extract and the mulberry leaf extract, concentrate to a clear paste with a relative density of 1.10-1.15, then add the turmeric oleoresin, concentrate and dry to obtain the composition.
[0013] In some specific embodiments, the raw materials of the composition include, by weight: 30 parts of turmeric, 30 parts of Ampelopsis grossedentata, and 10 parts of mulberry leaves.
[0014] In a second aspect, the present invention provides the application of the preparation method in preparing a feed or a drug for improving the ammonia nitrogen stress resistance ability of Micropterus salmoides. The ways for the feed or the drug to protect the fish body from ammonia nitrogen stress damage include at least one of the following (A1)-(A4):
[0015] (A1) Repairing the pathological damage of liver tissue;
[0016] (A2) Maintaining the normal function of gill filaments;
[0017] (A3) Regulating the oxidative stress level;
[0018] (A4) Maintaining osmotic pressure balance.
[0019] In a third aspect, the present invention provides a composition for improving the ammonia nitrogen stress resistance ability of Micropterus salmoides, prepared according to the preparation method.
[0020] Fourth aspect, the present invention provides an application of the composition in preparing a feed for improving the ability of largemouth bass to resist ammonia nitrogen stress.
[0021] In some specific embodiments, the composition for improving the ability of largemouth bass to resist ammonia nitrogen stress is added to the basic feed. Further, the addition amount of the composition is 1% of the weight of the basic feed.
[0022] Fifth aspect, the present invention provides a feed for improving the ability of largemouth bass to resist ammonia nitrogen stress, which comprises a basic feed and the composition for improving the ability of largemouth bass to resist ammonia nitrogen stress. Further, the addition amount of the composition is 1% of the weight of the basic feed.
[0023] The beneficial effects of the present invention at least include the following:
[0024] The composition provided by the present invention shows the ability to effectively reduce the mortality rate of largemouth bass under the condition of acute stress of high-concentration ammonia nitrogen (the ammonia nitrogen concentration in the water body is 20.15 mg / L), and shows significant advantages in improving the survival ability of fish. After applying the composition, the Na + / K + -ATPase activity in the gill filaments of largemouth bass is significantly increased, thereby effectively maintaining the osmotic pressure balance of fish. At the same time, the cortisol and lactic acid contents in the serum are significantly reduced, and this change indicates that the composition helps to relieve the stress response of largemouth bass caused by ammonia nitrogen stress and improves the energy metabolism state of fish.
[0025] The experimental results also show that under chronic long-term stress of low-concentration ammonia nitrogen (the ammonia nitrogen concentration in the water body is 4 mg / L), hematological indexes such as red blood cell count, hemoglobin count and white blood cell count are also improved, indicating a possible enhancement of fish immune function, which is of great significance for fish to resist external environmental pressure and pathogen invasion. By detecting serum biochemical indexes, the present invention further finds that the application of the composition helps to maintain the metabolic balance in the body of largemouth bass and significantly reduces the metabolic disorders caused by chronic ammonia nitrogen stress, which is crucial for ensuring the normal growth and development of fish. The results of histological pathological section observation show that the composition can significantly reduce the damage of chronic ammonia nitrogen stress to the liver tissue of largemouth bass and effectively protect the normal structure and function of liver cells. Therefore, the present invention not only has great potential in protecting important organs of fish, but also provides new ideas for solving fish health problems caused by ammonia nitrogen stress.
[0026] The present invention also determines the optimal ratio of turmeric, Ampelopsis grossedentata and mulberry leaves as raw materials of the composition, which not only fully realizes the significant synergistic effect among the components, but also ensures the good comprehensive benefits shown by the composition in aspects such as reducing mortality, increasing enzyme activity, alleviating stress response, reducing tissue damage, enhancing stress resistance ability and protecting the liver. Therefore, it has broad application prospects and market value. BRIEF DESCRIPTION OF THE DRAWINGS
[0027] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.
[0028] Figure 1 For the embodiment of the present invention, it shows the change of Na + / K + -ATPase activity in the gills of Micropterus salmoides under acute ammonia nitrogen stress; the abscissa represents the duration of ammonia nitrogen stress (24h, 48h, 72h and 96h); if the bar graphs at the same time point are marked with different lowercase letters (such as a, b, c), it indicates that there are significant differences between different treatment groups (P < 0.05, the same below);
[0029] Figure 2 For the embodiment of the present invention, it shows the change of cortisol content in the serum of Micropterus salmoides under acute ammonia nitrogen stress;
[0030] Figure 3 For the embodiment of the present invention, it shows the change of lactic acid content in the serum of Micropterus salmoides under acute ammonia nitrogen stress;
[0031] Figure 4 For the embodiment of the present invention, it is a pathological section of the liver tissue of Micropterus salmoides (HE staining, 200 times); among them: the arrow "→" indicates the hepatic sinusoid; It indicates the disorder of hepatic tissue structure and unclear cell boundaries;
[0032] Figure 4 a is the normal control group;
[0033] Figure 4 b is the medicated group;
[0034] Figure 4 c is the negative control group. DETAILED DESCRIPTION OF THE EMBODIMENTS
[0035] To make the purpose, technical scheme and advantages of the present invention clearer, the technical scheme in the present invention will be clearly and completely described below. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection of the present invention. If the specific conditions are not specified in the embodiments, they are carried out according to the normal conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified in the reagents or instruments used, they are all conventional products that can be purchased commercially.
[0036] Example 1 Ammonia nitrogen acute stress test
[0037] 1. Preparation of compound preparations
[0038] The preparation process of compound preparation 1-5 (hereinafter referred to as compound preparation) is as follows:
[0039] (1) Preparation of turmeric oleoresin:
[0040] Weigh 30.0g of turmeric, add 8 times its volume (240mL) of 85% (v / v) ethanol solution, and perform reflux extraction 3 times, each time for 2h. After the extraction is completed, the obtained extracts are combined and concentrated under reduced pressure to make 3g of thick paste. Subsequently, 30mL of 70% (v / v) ethanol is added to the thick paste and allowed to stand at 4°C for 24h for crystallization. Take the supernatant and continue to concentrate to a relative density of 1.13 (measured at 50°C) to obtain oleoresin, which is turmeric oleoresin.
[0041] (2) Preparation of rattan tea extract:
[0042] Weigh 30.0 g of rattan tea, add 8 times volume (240 mL) of 85% (v / v) ethanol solution, and perform reflux extraction 3 times, each time for 2 hours. After the extraction is completed, combine the obtained extracts to obtain rattan tea extract.
[0043] (3) Preparation of mulberry leaf extract:
[0044] Weigh 10.0 g of mulberry leaves, add 10 times the volume (100.0 mL) of deionized water, place in a boiling water bath, and perform 3 extractions, each for 2 hours. After the extraction is completed, combine the extracts to obtain a mulberry leaf extract.
[0045] (4) Preparation of compound preparations:
[0046] After mixing the rattan tea extract and the mulberry leaf extract, the mixture is concentrated under reduced pressure to prepare a clear paste (measured at 50° C., the relative density of the clear paste is 1.10). Turmeric oleoresin is added to the clear paste and stirred thoroughly to homogenize. Finally, the mixture is concentrated and dried to obtain the final compound preparation powder.
[0047] (5) The raw material ratios of curcuma oil resin, Ampelopsis grossedentata extract and mulberry leaf extract in Compound preparations 1-5 are as follows (by weight):
[0048] Compound 1: 30 parts of curcuma, 30 parts of Ampelopsis grossedentata, 10 parts of mulberry leaf;
[0049] Compound 2: 30 parts of curcuma, 30 parts of Ampelopsis grossedentata, 30 parts of mulberry leaf;
[0050] Compound 3: 30 parts of curcuma, 30 parts of Ampelopsis grossedentata, 20 parts of mulberry leaf;
[0051] Compound 4: 30 parts of curcuma, 20 parts of Ampelopsis grossedentata, 10 parts of mulberry leaf;
[0052] Compound 5: 30 parts of curcuma, 20 parts of Ampelopsis grossedentata, 20 parts of mulberry leaf.
[0053] 2. Experimental animals and breeding management
[0054] The initial body weight of the largemouth bass (Micropterus salmoides), the experimental fish used in this application, was (18.20 ± 0.81 g). They were purchased from Hubei Weilicheng Agricultural Technology Co., Ltd. Before the start of the experiment, they were temporarily raised with a basic diet (protein content 48% m / m, purchased from Tongwei Co., Ltd., Cat.No.: 20240305) for one week. The experimental fish were randomly divided into 7 groups, one normal control group (not fed medicine, not stressed), one negative control group (not fed medicine, stressed), and 5 medicated groups (fed medicine, stressed). Each group had 3 replicates, with 30 fish in each replicate. They were placed in a glass breeding system (57.8 cm × 38.2 cm × 22.7 cm). During the experiment, they were fed to satiation at 8:30 - 9:00 in the morning and 17:00 - 18:00 in the afternoon every day. During the experiment, the water temperature was maintained at 26 ± 2 °C, the dissolved oxygen was about 6 mg / L, the pH value was 6.8 ± 0.3, and the ammonia nitrogen was < 0.05 mg / L. The feces in the breeding tank were cleaned every afternoon, and the filter pool, filter screen, and filter stones were cleaned every three days, and 1 / 3 of the water volume in the breeding system was replaced.
[0055] 3. Experimental design
[0056] The medicine was administered by mixing it with the feed. The addition amount of the compound preparation in the feed was 1% (m / m) respectively. The normal control group and the negative control group were fed the basic feed, and the medicated groups were fed the medicated feed containing 1% (m / m) of Compound preparations 1-5 by mass ratio, and fed continuously for 30 days.
[0057] After 30 days of feeding all the experimental fish, they were starved for 24 h to adapt to the environment. The negative control group and the medicated groups were subjected to 96 h ammonia nitrogen stress, and the normal control group was observed synchronously without stress. No food was fed during the stress period for all groups. The median lethal concentration LC of ammonia nitrogen on Micropterus salmonides for 96 h was measured through a preliminary experiment 50The concentration was 20.15 mg / L, so 20.15 mg / L was taken as the stress concentration of ammonia nitrogen. NH4Cl solution was added to the negative control tank and the medicated tank to make the ammonia nitrogen concentration in the water body 20.15 mg / L, and an ammonia nitrogen stress test was carried out for 96 h, and the death situation of the test fish was recorded.
[0058] 4. Na + / K + Determination of Na
[0059] At 24 h, 48 h, 72 h, and 96 h of the anti-ammonia nitrogen stress test, 5 fish were taken from each parallel, blood was taken from the caudal vein, left to stand overnight at 4 °C, centrifuged at 3000 r / min for 10 min, and the supernatant was stored at -80 °C for later use. At the same time, the gills were separated. The serum determination indexes were cortisol and lactic acid, and the gill determination index was Na + / K + -ATPase, and the determination was carried out according to the method of the kit; cortisol (Cat.No.: 240402), lactic acid (Cat.No.: 20240316), and Na + / K + -ATPase (Cat.No.: 20240418) detection kits were all purchased from Nanjing Jiancheng Bioengineering Institute. The data were all expressed as "mean ± standard deviation", and Excel 2017 and SPSS19.0 statistical software were used for drawing and data analysis.
[0060] 5. Results
[0061] (1) The death results are statistically as follows:
[0062] Table 1 Mortality of Micropterus salmoides caused by acute ammonia nitrogen stress (96 h ammonia nitrogen stress)
[0063]
[0064]
[0065] Under acute ammonia nitrogen stress conditions, specific behavioral responses and physiological state changes were observed in Micropterus salmoides, specifically manifested as follows: the test fish swam around the wall of the experimental barrel. As the exposure time to ammonia nitrogen toxicity increased, the fish gradually showed spastic convulsion symptoms, turned upside down with the abdomen facing up, and the overall vitality decreased significantly, showing an abnormal swimming pattern of sometimes diving to the bottom and sometimes jumping out of the water sharply. After this state lasted for about 4 - 6 min, the fish body finally slowly sank to the bottom, with the mouth and gills wide open until death. The mortality rate in the negative control group was as high as 53.33% within 96 h.
[0066] In contrast, among the groups treated with the compound preparations, the mortality rate of the fish showed a decreasing trend to varying degrees. Notably, Group 1 of the compound preparation exhibited the lowest mortality rate, only 18.89%. This result indicates that the formulation of Compound Preparation 1 (the formulation refers to the reasonable combination of two or more drugs according to pathological needs and drug characteristics, aiming to enhance the drug effect, reduce toxicity or broaden the treatment spectrum) demonstrated the optimal effect and could effectively reduce the fish mortality caused by acute ammonia nitrogen stress.
[0067] (2) Effects of compound preparations on the Na + / K + -ATPase activity in the gill filaments of Micropterus salmoides under acute ammonia nitrogen stress
[0068] Acute ammonia nitrogen stress showed a significant inhibitory effect on the Na + / K + -ATPase activity (P < 0.05). At each sampling time point, the enzyme activities of all groups exposed to ammonia nitrogen stress (including the negative control group and the compound preparation treatment groups) were significantly lower than those of the normal control group. This result is consistent with previous studies, further confirming that ammonia nitrogen stress can interfere with the fish body's osmotic regulation mechanism, affect the functions of chloride-secreting cells and protease on the organelle membrane in the gills, and thus disrupt the osmotic pressure balance of the fish body.
[0069] As Figure 1 the results show, when treating Micropterus salmoides under ammonia nitrogen stress with Compound Preparations 1, 2, and 3, a significant increase in enzyme activity was shown (P < 0.05). Compared with the negative control group, these compound preparations could effectively reduce the adverse effects of ammonia nitrogen stress on the Na + / K + -ATPase activity and maintain the relative stability of the gill filament regulation function. Among them, Compound Preparation 1 performed the best, and it is speculated that its component ratio or the synergistic effect between components may be more conducive to alleviating the damage of ammonia nitrogen stress to the fish body. In contrast, the increase in enzyme activity of Compound Preparations 4 and 5 in the treatment groups was not significant (P > 0.05), indicating that these two compound preparations have limited effects in alleviating the inhibition of ammonia nitrogen stress on enzyme activity. This may be related to the fact that the formulation components or ratios failed to effectively counteract the effects of ammonia nitrogen stress.
[0070] From the changes in enzyme activity over time within all groups, it can be seen that the enzyme activity showed a slight increase at 48 h, and then gradually decreased, and there was no significant difference in enzyme activity at 72 h and 96 h (P > 0.05). This change pattern may reflect the adaptive response of the fish body in the initial stage of ammonia nitrogen stress, that is, by activating the internal osmotic mechanism and attempting to increase enzyme activity through active osmotic regulation. However, with the continuous damage of ammonia nitrogen to the gill tissue, the Na + / K +-ATPase activity gradually decreased and reached a relatively stable low level under long-term stress, indicating that when the fish was exposed to ammonia nitrogen stress for a long time, the recovery or adaptation of its Na + / K + -ATPase activity had limitations, highlighting the persistent and irreversible damage of acute ammonia nitrogen stress to the fish.
[0071] (3) Effects of compound preparations on the serum cortisol content of Micropterus salmoides under acute ammonia nitrogen stress
[0072] It can be seen from Figure 2 that ammonia nitrogen stress significantly increased the cortisol content in the serum of Micropterus salmoides (P < 0.05). At each sampling time point, the cortisol content in all groups exposed to ammonia nitrogen stress (including the negative control group and the compound preparation treatment groups) was significantly higher than that in the normal control group. Research has shown that cortisol levels in fish increase under stress conditions, and the severity of stress can be indicated by the degree and duration of this increase. This result shows that acute ammonia nitrogen stress can cause Micropterus salmoides to be in an acute stress state, and the stress state lasts until 96 h. From the change of cortisol content within each group over time, it can be seen that the cortisol content had a slight upward trend after 48 h, and the increase in cortisol content was not significant after 72 h, with no significant difference compared with the value at 96 h (P > 0.05). Acute ammonia nitrogen stress caused a stress response in Micropterus salmoides, and the cortisol in the blood increased rapidly, reaching a peak at 72 h and then tending to be stable.
[0073] At the same time point, the cortisol content in the serum of Compound 1 and Compound 2 was significantly lower than that in the negative control group (P < 0.05), indicating that Compound 1 and Compound 2 could well alleviate the stress response under acute ammonia nitrogen stress. Among them, Compound 1 had the best effect, and it was speculated that its component ratio or the synergistic effect between components might be more beneficial to relieve the fish stress caused by ammonia nitrogen stress. In contrast, the alleviating effects of Compound Preparations 3, 4, and 5 in the treatment groups were not significant (P > 0.05), indicating that these three compound preparations had limited effects in relieving the fish stress caused by ammonia nitrogen stress. This might be related to the fact that the preparation components or ratios failed to effectively counteract the effects of ammonia nitrogen stress.
[0074] (4) Effects of compound preparations on the serum lactic acid content of Micropterus salmoides under ammonia nitrogen stress
[0075] It can be seen from Figure 3It can be seen that acute ammonia nitrogen stress significantly increased the lactic acid content in the serum of Micropterus salmoides (P < 0.05). At each sampling time point, the lactic acid content in all groups exposed to ammonia nitrogen stress (including the negative control group and the compound preparation treatment group) was significantly higher than that in the normal control group. Lactic acid is a product of anaerobic respiration energy metabolism and can reflect the mode of respiratory metabolism. When fish are stressed by ammonia nitrogen, the gill tissue is damaged to a certain extent, affecting gas exchange, and then causing the fish body to be hypoxic. The aerobic metabolism of the body is blocked and anaerobic metabolism is initiated, so the lactic acid content in the blood increases. This result indicates that acute ammonia nitrogen stress will cause certain damage to the gill filament tissue of Micropterus salmoides. From the change of cortisol content within each group over time, it can be seen that starting from 48 h, the lactic acid in the serum of Micropterus salmoides showed an upward trend, and the increase in lactic acid content was not significant after 72 h, showing no significant difference compared with the value at 96 h (P > 0.05). It shows that acute ammonia nitrogen stress causes damage to the gill filaments of Micropterus salmoides, and the resulting impact on gas exchange in Micropterus salmoides will continue until 96 h.
[0076] At the same time point, Compound 1 - 3 could significantly reduce the increase in lactic acid content in the serum (P < 0.05). Compared with the negative control group, these compound preparations could effectively reduce the adverse effects of ammonia nitrogen stress on the gill filaments and maintain the relative stability of the gas exchange function of the gill filaments. Compound 1 had the best effect, and it was speculated that its component ratio or the synergistic effect between components might be more conducive to alleviating the damage of fish gill filaments caused by ammonia nitrogen stress. In contrast, the alleviating effects of Compound Preparations 3, 4, and 5 in the treatment groups were not significant (P > 0.05), indicating that these three compound preparations had limited effects in alleviating the damage of fish gill filaments caused by ammonia nitrogen stress.
[0077] Example 2 Anti - chronic ammonia nitrogen stress test
[0078] 1. Preparation of compound preparation
[0079] The preparation process of the compound preparation (hereinafter referred to as compound for short) is as follows:
[0080] (1) Preparation of turmeric oleoresin:
[0081] Weigh 30.0 g of turmeric, add 8 - fold volume (240 mL) of 85% (v / v) ethanol solution, and perform reflux extraction 3 times, each time lasting 2 h. After the extraction is completed, combine the obtained extraction solutions and perform vacuum concentration to make a thick paste of 3 g. Subsequently, add 30 mL of 70% (v / v) ethanol to the thick paste and let it stand for crystallization at 4℃ for 24 h. Take the supernatant and continue to concentrate it to a relative density of 1.13 (measured at 50℃) to obtain the oleoresin, which is turmeric oleoresin.
[0082] (2) Preparation of Ampelopsis grossedentata extract:
[0083] Weigh 30.0 g of Ampelopsis grossedentata, add 8-fold volume (240 mL) of 85% (v / v) ethanol solution, and perform reflux extraction 3 times, 2 h each time. After extraction, combine the obtained extracts to obtain the Ampelopsis grossedentata extract.
[0084] (3) Preparation of mulberry leaf extract:
[0085] Weigh 10.0 g of mulberry leaves, add 10-fold volume (100.0 mL) of deionized water. Place it in a boiling water bath environment and perform extraction 3 times, 2 h each time. After extraction, combine the extracts to obtain the mulberry leaf extract.
[0086] (4) Preparation of the compound preparation:
[0087] Mix the Ampelopsis grossedentata extract and the mulberry leaf extract, then concentrate under reduced pressure to make a clear paste (measured at 50 °C, the relative density of the clear paste is 1.10). Add curcuma oil resin to the clear paste and stir well for homogenization. Finally, perform concentration and drying treatment to obtain the final compound preparation powder.
[0088] (5) The raw material ratios of curcuma oil resin, Ampelopsis grossedentata extract and mulberry leaf extract in the compound preparation are as follows (by weight): 30 parts of curcuma, 30 parts of Ampelopsis grossedentata, and 10 parts of mulberry leaves.
[0089] 2. Experimental animals and breeding management
[0090] The initial body weight of the largemouth bass (Micropterus salmoides) used in this application is (18.20 ± 0.81 g). It is purchased from Hubei Weilicheng Agricultural Technology Co., Ltd. Before the experiment starts, first acclimate with a basal diet (protein content 48% m / m, purchased from Tongwei Co., Ltd., Cat. No.: 20240507) for one week. Randomly divide the experimental fish into 7 groups, one normal control group (not medicated, not stressed), one negative control group (not medicated, stressed), and 5 medicated groups (medicated, stressed). Each group has 3 replicates, and each replicate has 30 fish. They are respectively placed in a glass culture system (57.8 cm × 38.2 cm × 22.7 cm). Feed them to satiation at 8:30 - 9:00 in the morning and 17:00 - 18:00 in the afternoon every day. During the experiment, the water temperature is maintained at 26 ± 2 °C, the dissolved oxygen is about 6 mg / L, the pH value is 6.8 ± 0.3, and the ammonia nitrogen is < 0.05 mg / L. Clean the feces in the culture tank every afternoon, clean the filter pool, filter screen and filter stones every three days, and change 1 / 3 of the water volume of the culture system.
[0091] 3. Experimental design
[0092] At the beginning of the experiment, add NH4Cl solution to the water bodies of the negative control group and the medicated groups to make the ammonia nitrogen concentration 4 mg / L (measured by preliminary experiment for 96 h LC 50It was 20.15 mg / L, and 20% of the safety concentration was taken as 4 mg / L. The ammonia nitrogen concentration in the water body was measured every other day and adjusted in a timely manner to maintain the ammonia nitrogen concentration at 4 mg / L throughout the test period (30 d). No NH4Cl solution was added to the water body of the normal control group.
[0093] During the test period, the drug was administered by mixing it with the feed. The addition amounts of the compound preparation in the feed were both 1% (m / m). The normal control group and the negative control group were fed the basal feed, and the medicated groups were fed the medicated feed containing 1% (m / m) of the compound preparation by mass for 30 consecutive days.
[0094] 4. Determination of hematological indexes and serum biochemical indexes
[0095] 24 h after the feeding test ended, 5 fish were randomly selected from each tank, and blood was taken from the caudal vein. Part of the blood was injected into an anticoagulation tube to prepare anticoagulated blood, which was stored at 4 °C for the determination of hematological indexes; the other part was injected into an EP tube, left standing at 4 °C overnight, centrifuged at 3000 r / min for 10 min, and the upper-layer serum was stored at -80 °C for the determination of serum biochemical indexes.
[0096] The hematological indexes were red blood cell count (RBC), white blood cell count (WBC), and hemoglobin (HGB), which were determined using a Mindray veterinary automatic blood cell analyzer (model: BC-2800vet).
[0097] The serum determination indexes were malondialdehyde (MDA) and superoxide dismutase (SOD), which were determined according to the kit method. The SOD kit (Cat. No.: 20240520) and the MDA kit (Cat. No.: 20240603) were purchased from Nanjing Jiancheng Reagent Company.
[0098] The data were all expressed as "mean ± standard deviation", and Excel 2017 and SPSS 19.0 statistical software were used for drawing and data analysis.
[0099] 5. Observation of liver tissue pathological sections
[0100] After blood collection, the livers of 3 fish were randomly taken from each tank, fixed with 4% neutral formaldehyde solution, paraffin sections were made, HE stained, and examined and photographed under a microscope.
[0101] 6. Experimental results
[0102] (1) Effects of the compound preparation on the hematological indexes of Micropterus salmoides under chronic ammonia nitrogen stress
[0103] As can be seen from Table 2, the number of red blood cells and hemoglobin in the negative control group and the medicated group were significantly lower than those in the normal control group (P < 0.05), and the number of red blood cells and hemoglobin in the medicated group were significantly higher than those in the negative control group (P < 0.05); the number of white blood cells in the negative control group was significantly lower than those in the normal control group and the medicated group (P < 0.05), and there was no significant difference between the normal control group and the medicated group (P > 0.05). Due to the long-term stress of ammonia nitrogen, the number of red blood cells, white blood cells, and hemoglobin in the test fish of the negative control group and the medicated group were all lower than those in the normal control group, and the indicators of the medicated group were higher than those of the negative control group. Among them, there was no significant difference in the number of white blood cells compared with the normal group, indicating that ammonia nitrogen stress affects the hematopoietic function and immune system of fish, resulting in changes in the number of red blood cells, white blood cells, and hemoglobin. The compound preparation alleviates the toxic effect of ammonia nitrogen, improves the immunity and metabolic function of fish, and thus relieves the damage of ammonia nitrogen stress to the fish body.
[0104] Table 2 Changes in hematological indicators
[0105] Group <![CDATA[Red blood cell count (×10 12 / L)]]> <![CDATA[White blood cell count (×10 10 / L)]]> Hemoglobin (×g / L) Normal control group <![CDATA[3.68±0.27 a > <![CDATA[4.54±0.24 a > <![CDATA[74.58±6.84 a > Negative control group <![CDATA[2.16±0.21 c > <![CDATA[3.51±0.31 b > <![CDATA[43.46±6.49 c > Drug treatment group <![CDATA[3.28±0.26 b > <![CDATA[4.03±0.30 a > <![CDATA[62.44±5.24 b >
[0106] Note: Different letters on the right shoulder of each column of data indicate significant differences (P < 0.05).
[0107] (2) Effects of compound preparation on serum biochemical indicators of largemouth bass under chronic ammonia nitrogen stress
[0108] As can be seen from Table 3, there were significant differences in the activity of Na + / K + -ATPase among the three groups (P < 0.05), with the normal control group > the medicated group > the negative control group. There were significant differences in serum cortisol and lactate among the three groups (P < 0.05), with the negative control group > the medicated group > the normal control group. Na + / K + -ATPase is a protease on the chloride-secreting cells and organelle membranes of the gills and mainly participates in the osmotic regulation process in fish. When the gill filament tissue is damaged, it will lead to a decrease in enzyme activity; when fish are stressed by ammonia nitrogen, the gill tissue is damaged to a certain extent, affecting gas exchange, and then causing the fish body to be hypoxic, and the aerobic metabolism of the body is blocked and anaerobic metabolism is initiated. Therefore, the lactate content in the blood increases; the cortisol level in fish increases under stress conditions, and the severity of stress can be indicated by the degree and duration of this increase. Therefore, the cortisol content can be an important physiological indicator of the stress level. From the results of Table 3, it can be seen that feeding the compound preparation can reduce the damage caused by long-term ammonia nitrogen stress to the gill filaments of largemouth bass and improve the ability of largemouth bass to resist ammonia nitrogen stress.
[0109] Table 3 Changes in serum biochemical indicators
[0110]
[0111] Note: Different letters on the right shoulder of each column of data indicate significant differences (P < 0.05).
[0112] (3) Histopathological sections
[0113] It can be seen from Figure 4 that in the normal control group ( Figure 4 a) The number of hepatic sinusoids is large and the hepatic sinusoids are filled, indicating that the blood flow in the liver tissue of the normal control group is large and the liver tissue develops well. In the medicated group ( Figure 4 b) The number of hepatic sinusoids is more than that in the negative control group ( Figure 4 c), but less than that in the normal control group; in the negative control group (c), the hepatocyte structure in some areas of the liver tissue is disordered and the boundary is not clear. Under the long-term stress of low-concentration ammonia nitrogen, the liver tissue of the negative control group and the medicated group is damaged to a certain extent and the development status is worse than that of the normal group. The compound preparation plays a certain protective role in the liver of Micropterus salmoides, and the liver tissue condition is better than that of the negative control group.
[0114] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.
Claims
1. A method for preparing a composition for improving the ability of largemouth bass to resist ammonia nitrogen stress, characterized in that: The steps include: (S1) adding 8 times the volume of 85% v / v ethanol solution to 28-32 parts by weight of turmeric, refluxing and extracting for 2-4 times, each time for 1.5-3 hours, collecting and concentrating the extract to obtain a thick paste; adding 1 times the volume of 70% v / v ethanol solution to the thick paste, crystallizing at 4° C., standing for 12-36 hours, collecting the supernatant, and concentrating to a relative density of 1.10-1.15 to obtain turmeric oleoresin; (S2) adding 8 times the volume of 85% v / v ethanol solution to 28-32 parts by weight of rattan tea, reflux extraction 2-4 times, each time for 1.5-3 hours, combining the extracts from each extraction to obtain a rattan tea extract; (S3) adding 10 times the volume of deionized water to 8-12 parts by weight of mulberry leaves, placing the mixture in a boiling water bath, and extracting the mixture 2-4 times, each time for 1.5-3 hours, and combining the extracts from each extraction to obtain a mulberry leaf extract; (S4) mixing the rattan tea extract and the mulberry leaf extract, concentrating to a clear paste with a relative density of 1.10-1.15, adding the turmeric oleoresin, concentrating and drying to obtain the composition.
2. The preparation method according to claim 1, characterized in that: The raw materials of the composition include, by weight, 30 parts of turmeric, 30 parts of rattan tea and 10 parts of mulberry leaves.
3. Use of the preparation method according to claim 1 or 2 in preparing feed or medicine for improving the ability of largemouth bass to resist ammonia nitrogen stress.
4. The use according to claim 3, characterized in that: The feed or drug protects fish from damage caused by ammonia nitrogen stress by at least one of the following (A1)-(A4): (A1) repairing pathological damage to liver tissue; (A2) maintaining the normal function of gill filaments; (A3) Regulate oxidative stress levels; (A4) Maintain osmotic pressure balance.
5. A composition for improving the ability of largemouth bass to resist ammonia nitrogen stress, characterized in that: Prepared according to the preparation method of claim 1 or 2.
6. Use of the composition according to claim 5 in preparing a feed for improving the ability of largemouth bass to resist ammonia nitrogen stress.
7. The use according to claim 6, characterized in that: The composition for improving the ability of largemouth bass to resist ammonia nitrogen stress is added into the basic feed.
8. The use according to claim 7, characterized in that: The addition amount of the composition for improving the ability of largemouth bass to resist ammonia nitrogen stress is 1% of the weight of the basic feed.
9. A feed for improving the ability of largemouth bass to resist ammonia nitrogen stress, characterized in that: The invention comprises a basic feed and the composition as claimed in claim 3.
10. The feed according to claim 9, characterized in that Calculated by weight, the composition for improving the ability of largemouth bass to resist ammonia nitrogen stress is added in an amount of 1% of the weight of the basic feed.