Method for evaluating ammonia nitrogen stress protection effect of fish feed raw material

Abstract

The invention discloses a method for screening ammonia nitrogen stress protection effects of fish feed raw materials. The screening method comprises the following steps: S1, mixing a feed raw material to be evaluated with a basal feed, granulating, preparing a test feed, and feeding zebra fish; and S2, after fasting, transferring the zebrafish into an ammonium chloride solution for acute ammonia nitrogen stress, observing and recording death time until all the zebrafish die, and recording a survival curve of the zebrafish. According to the method provided by the invention, researchers and feed enterprises can objectively compare the relieving capacities of different raw materials on ammonia nitrogen stress. According to the invention, high efficiency and repeatability of the evaluation process are realized. The zebra fish model is low in cost and easy to operate, and experimental variable interference is reduced by combining a basal feed formula; according to the method, remarkable economic benefits and environmental benefits are brought, through index ranking, enterprises can preferentially select raw materials with high protection effects, the survival rate of fishes is increased, the culture cost is reduced, ammonia nitrogen stress can be dealt with, and the aquaculture industry is promoted to develop towards the green and intensive direction.

Description

Technical Field

[0001] This invention relates to a method and application for screening the protective effect of ammonia nitrogen stress in fish feed ingredients, belonging to the field of aquatic animal feed ingredients. Background Technology

[0002] As global aquaculture develops towards intensification and high density, the decomposition of organic matter such as uneaten feed and feces leads to a significant increase in ammonia nitrogen concentration in water bodies. The accumulation of ammonia nitrogen in water has become one of the key environmental factors restricting the sustainable development of aquaculture. Ammonia nitrogen in water mainly enters the fish body in the form of NH3 through the gills and body surface. Ammonia nitrogen poisoning can cause fish to experience decreased growth rate, weakened immune levels, and oxidative stress and inflammatory responses, which can lead to fish mortality in severe cases.

[0003] The protein levels and amino acid composition of different feed ingredients directly affect the amount of ammonia nitrogen produced by fish metabolism. Excessive protein content in feed ingredients (such as fishmeal and soybean meal) or an imbalanced amino acid profile (such as miscellaneous meal feed ingredients) may increase ammonia nitrogen excretion and lead to nitrogen waste.

[0004] Feed ingredients contain various substances with antioxidant activity, such as vitamin C, vitamin E, carotenoids, and beneficial components like polysaccharides. These nutrients can participate in non-enzymatic antioxidant reactions in fish, helping them cope with ammonia nitrogen stress. Therefore, studying the protective ability of different feed ingredients against ammonia nitrogen stress in fish is of great significance. Summary of the Invention

[0005] The purpose of this invention is to overcome the gaps in existing research and provide a method for evaluating the protective effect of ammonia nitrogen stress in fish feed ingredients. This method can be applied to the evaluation of actual feed ingredients to achieve a quantitative comparison of the protective effect.

[0006] The method for screening the protective effect of ammonia nitrogen stress on fish feed ingredients provided by the present invention includes the following steps:

[0007] S1. The feed ingredients to be evaluated are mixed with the basic feed and pelleted to prepare the experimental feed, which is then fed to zebrafish.

[0008] S2. After fasting, the zebrafish were transferred to an ammonium chloride solution to undergo acute ammonia nitrogen stress. The time of death was observed and recorded until all fish died, and the zebrafish survival curve was recorded.

[0009] Preferably, the concentration of the ammonium chloride solution is 40 mg / L, which causes complete death of zebrafish within 48 hours, meeting the requirements for acute stress and laying the foundation for method standardization.

[0010] In the method of the present invention, the feed raw materials include fish meal or soybean meal, which are crushed and passed through a 60-mesh sieve and then mixed with the basic feed to form pellets.

[0011] Preferably, the basic feed includes casein, gelatin, dextrin, soybean oil, lysine, vitamin C, multivitamins, multiminerals, calcium dihydrogen phosphate, choline, sodium alginate, and zeolite powder.

[0012] The mass ratio of the feed ingredients to the basic feed is 1-4:9-6, preferably 3:7.

[0013] In the screening method of this invention, the initial weight of the zebrafish is (30-60)±5mg;

[0014] The feeding period is 10-14 days;

[0015] The fasting period is 24-48 hours.

[0016] The conditions for acute ammonia nitrogen stress are as follows:

[0017] The aquaculture water temperature is 26-28℃, the dissolved oxygen is 6-8mg / L, and the ammonium chloride solution is changed every 12-24 hours, maintaining an 8-12 hour light / dark cycle.

[0018] The method of the present invention further includes the step of determining the ammonia nitrogen stress resistance index based on the zebrafish survival curve, so as to evaluate the protective effect of the feed ingredient against ammonia nitrogen stress.

[0019] Ammonia nitrogen stress resistance index = B / A

[0020] B represents the limited mean survival time of the experimental group, and A represents the limited mean survival time of the control group.

[0021] The restricted average survival time is obtained based on the zebrafish survival curves over the entire experimental period, which is defined as the time period from the occurrence of deaths in all groups to the death of the last zebrafish.

[0022] The restricted mean survival time (RMS) is an important indicator in survival analysis. It considers the average survival time within a specific time constraint and can more comprehensively reflect the characteristics of the survival curve. Calculating the survival curve difference based on RMS allows for a more flexible consideration of survival situations at different time points, especially when survival curves intersect or the disproportionate risk assumption does not hold, providing more accurate comparison results.

[0023] The higher the value of the anti-ammonia nitrogen stress index, the better the protective effect.

[0024] This invention provides a standardized method that enables researchers and feed companies to objectively compare the ability of different feed ingredients to alleviate ammonia nitrogen stress. This invention achieves high efficiency and reproducibility in the evaluation process. The zebrafish model is low-cost and easy to operate, and combined with basic feed formulations, it reduces interference from experimental variables. For example, this invention significantly improves experimental efficiency by grouping experimental feeds with different feed ingredients (such as fishmeal and soybean meal) with three replicates per group. This invention provides a high-precision quantitative tool that allows direct comparison of the protective effects of different feed ingredients; a larger index value (e.g., 2.13 for fishmeal group A) indicates a better resistance to ammonia nitrogen stress. This avoids subjective judgment and achieves objective ranking. This invention enhances the reliability and practicality of the method. Low dispersion (e.g., CV = 3.21% for fishmeal group E and CV = 5.44% for fishmeal group F) demonstrates the reproducibility of the results, avoids single-experiment errors, and is suitable for quality control and large-scale screening in feed companies. This invention brings significant economic and environmental benefits. By using index ranking, companies can prioritize raw materials with high protective effects (such as fishmeal with an index > 1.5), improve fish survival rates, reduce breeding costs, help cope with ammonia nitrogen stress, and promote the development of aquaculture towards green and intensive practices. Attached Figure Description

[0025] Figure 1 This is for screening the ammonium chloride concentration in zebrafish under acute ammonia nitrogen stress in Example 1 of the present invention;

[0026] Figure 2 The following are the ammonia nitrogen stress survival curves of zebrafish using different feed ingredients in Example 2 of this invention;

[0027] Figure 3 The survival curves are the results of three replicates of the same feed ingredient in Example 3 of this invention;

[0028] Figure 4 To ensure the repeatability of the ammonia nitrogen stress index assessment results of different feed ingredients based on limited mean survival time in Example 4 of this invention. Detailed Implementation

[0029] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0030] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0031] The basic feed ingredients, proportions, and nutritional levels for zebrafish in the following examples are shown in Table 1:

[0032] Table 1. Composition and Proportion of Basic Feed Ingredients

[0033] Ingredients (g / 100g diet) Basic feed Casein 40.00 gelatin 10.00 dextrin 28.00 Soybean oil 16.00 Lysine 0.33 Vitamin C 0.10 Multidimensional 0.40 Multiple minerals 0.40 calcium dihydrogen phosphate 2.00 choline 0.20 Sodium alginate 2.00 microcrystalline cellulose 0.00 Zeolite powder 0.57 total 100.00 Nutritional level (dry matter) Crude protein (%, calculated value) 42.19 Crude fat (%, calculated value) 16.09 Total energy (kJ / g, calculated value) 18.84

[0034] Example 1: Screening of ammonium chloride concentration in zebrafish under acute ammonia nitrogen stress

[0035] One hundred and forty healthy zebrafish juveniles of uniform size, weighing (40±5) mg, were randomly selected and temporarily raised on a basal diet. After two weeks of temporary rearing, all zebrafish were starved for 24 hours and then weighed. After weighing, the temporarily raised zebrafish were randomly divided into seven groups of 20 each. The seven groups were placed in aqueous solutions of ammonium chloride with concentrations of 20, 30, 40, 50, 60, 70, and 80 mg / L, respectively, to screen for ammonia nitrogen stress. The ammonia nitrogen solution was changed every 24 hours, and a 12-hour light-12-hour dark cycle was maintained during the ammonia nitrogen stress period. The zebrafish were fasted during the experiment, and mortality was recorded every 2 hours, and survival curves were plotted. Figure 1 As shown.

[0036] The zebrafish used in this invention were provided by the Feed Research Institute of the Chinese Academy of Agricultural Sciences, and all were AB strains.

[0037] Figure 1 The results showed that zebrafish died completely within 20 hours in ammonium chloride solutions of 50 mg / L, 60 mg / L, 70 mg / L, and 80 mg / L; in a 30 mg / L ammonium chloride solution, the survival rate of zebrafish reached 50% at 110 hours; and in a 20 mg / L ammonium chloride solution, the mortality rate was only 30% at 110 hours. Based on the results above, 40 mg / L ammonium chloride solution was selected as the concentration for ammonia stress in zebrafish.

[0038] Example 2: Using zebrafish as a model, the survival curves of ammonia nitrogen stress differed among different feed ingredients. Fishmeal and soybean meal were used as examples.

[0039] The feed ingredients, taking fishmeal and soybean meal as examples, were all pulverized and passed through a 60-mesh sieve. Then, the feed ingredients were mixed with the basal feed (Table 1) at a ratio of 3:7 and pelleted to prepare experimental feeds, namely the basal feed control group, fishmeal A treatment group, fishmeal B treatment group, fishmeal C treatment group, fishmeal D treatment group, soybean meal A treatment group, soybean meal B treatment group, and soybean meal C treatment group. The fishmeal and soybean meal used in this example were all from major domestic aquatic feed companies.

[0040] Table 2. Feed composition and ratio for each experiment

[0041] Table of contents Crude moisture % Crude ash content % Crude fat percentage Crude protein % Fishmeal A 9.56 16.85 7.27 65.98 Fish meal B 7.62 18.62 10.89 62.17 Fishmeal C 9.03 17.8 5.99 65.49 Fishmeal D 7.82 16.3 9.29 66.06 Fishmeal E 10 17.51 5.54 64.38 Fishmeal F 7.37 20.29 10.11 62.09 Soybean Meal A 11.64 6.11 1.2 45.46 Soybean Meal B 11.8 5.88 0.51 44.31 Soybean Meal C 12.10 6.07 0.20 44.72

[0042] Two hundred and seventy zebrafish with an initial body weight of 40 ± 5 mg were randomly divided into nine groups, with three replicates per group and ten fish per replicate. The control group was fed a basal diet, while the treatment groups were fed fishmeal and soybean meal, respectively. The experiment lasted for 14 days, and the feeding amount was recorded daily.

[0043] After the experiment, the zebrafish in each group were fasted for 24 hours and then weighed. After 24 hours of weighing, the zebrafish in each group were transferred to a pre-aerated 40 mg / L ammonium chloride solution to induce acute ammonia nitrogen stress. The water was changed every 24 hours during the experiment, and dead fish were removed periodically. Throughout the stress experiment, the water temperature was maintained at 27.5℃ and dissolved oxygen was >6.5 mg / L.

[0044] This embodiment records mortality rates in real time, improving counting accuracy. During stress, the time of death for each animal is accurately recorded when the zebrafish are in a state of death. A zebrafish survival curve is plotted with time on the x-axis and the cumulative survival function on the y-axis.

[0045] Statistical Analysis: This example uses Graphpad 9.0.0 to plot survival curves and perform statistical analysis. The results are as follows: Figure 2 As shown.

[0046] Figure 2 The results show that zebrafish fed with different fishmeal and soybean meal feed ingredients exhibit different ammonia nitrogen stress survival curves.

[0047] Example 3: Assessment of ammonia nitrogen stress resistance index of feed ingredients based on restricted mean survival time

[0048] Limiting mean survival time is defined as the area under the survival curve over a certain period of time, i.e., the average survival time. The longer the mean survival time, the better the treatment effect.

[0049] The relative effect can be measured by the ratio of the limited mean survival time of the experimental group to that of the control group.

[0050] This embodiment is based on the comparison of differences in survival curves with small samples and short periods. It calculates the restricted mean survival time of different feed ingredients throughout the entire life cycle, and uses the ratio of the restricted mean survival time of the experimental group to that of the control group as a measure of the difference in effect between the two groups, and describes it quantitatively.

[0051] In this experiment, the timeline was set from the occurrence of mortality in all groups to the death of the last zebrafish in any experimental group, constituting the entire experimental period. Throughout the experimental period, the mean survival time (MSS) limited by ammonia nitrogen stress was calculated as A for the control group and B for the experimental group. The ratio B / A was used as the ammonia nitrogen stress resistance index of the feed ingredients. The calculation formula is as follows:

[0052] The ammonia nitrogen stress index of a certain feed ingredient is calculated as B / A, where a larger index value indicates a better resistance to ammonia nitrogen stress.

[0053] Survival analysis was conducted on two types of raw materials: fishmeal and soybean meal. The average survival time and ammonia nitrogen stress resistance index were calculated using the formulations in Table 2 as controls. The ammonia nitrogen stress resistance index of fishmeal ranged from 0.62 to 2.13, while that of soybean meal ranged from 0.77 to 1.50.

[0054] Table 3. Anti-ammonia nitrogen stress index of different feed ingredients

[0055]

[0056] Example 4: Stability Validation of Feed Ingredients' Ammonia Nitrogen Stress Index Based on Limiting Mean Survival Time

[0057] Based on Table 2, different batches of control feed were prepared, and the stability analysis of the ammonia nitrogen stress index results among different batches of the same feed was conducted. The results are as follows: Figure 3 As shown and Figure 4 As shown.

[0058] The dispersion of the ammonia nitrogen stress index of different feed ingredients (dispersion = standard deviation / mean) is shown in Table 3.

[0059] Table 4. Dispersion of ammonia nitrogen stress index for different feed ingredients

[0060]

[0061] The results show that the ammonia nitrogen stress resistance index of different feed ingredients was assessed based on the limitation of mean survival time, and the results showed good repeatability of the ammonia nitrogen stress resistance index of different feed ingredients.

Claims

1. A method for screening the protective effect of ammonia nitrogen stress on fish feed ingredients, comprising the following steps: S1. The feed ingredients to be evaluated are mixed with the basic feed and pelleted to prepare the experimental feed, which is then fed to zebrafish. S2. After fasting, the zebrafish were transferred to an ammonium chloride solution to undergo acute ammonia nitrogen stress. The time of death was observed and recorded until all fish died, and the zebrafish survival curve was recorded.

2. The screening method according to claim 1, characterized in that: The concentration of the ammonium chloride solution is 40 mg / L.

3. The screening method according to claim 1 or 2, characterized in that: The feed ingredients include fish meal or soybean meal, which are crushed through a 60-mesh sieve and then mixed with the basic feed.

4. The screening method according to any one of claims 1-3, characterized in that: The basic feed includes casein, gelatin, dextrin, soybean oil, lysine, vitamin C, multivitamins, minerals, calcium dihydrogen phosphate, choline, sodium alginate, and zeolite powder. The mass ratio of the feed ingredients to the basal feed is 1-4:9-6.

5. The screening method according to any one of claims 1-4, characterized in that: The initial weight of the zebrafish was (30-60) ± 5 mg; The feeding period is 10-14 days; The fasting period is 24-48 hours.

6. The screening method according to any one of claims 1-5, characterized in that: The conditions for acute ammonia nitrogen stress are as follows: The aquaculture water temperature is 26-28℃, dissolved oxygen is 6-8mg / L, and the ammonium chloride solution is changed every 12-24 hours, maintaining an 8-12 hour light / dark cycle.

7. The screening method according to any one of claims 1-6, characterized in that: The method further includes a step of determining the ammonia nitrogen stress resistance index based on the zebrafish survival curve, in order to evaluate the protective effect of the feed ingredient against ammonia nitrogen stress. Ammonia nitrogen stress resistance index = B / A B represents the limited mean survival time of the experimental group, and A represents the limited mean survival time of the control group. The restricted average survival time is obtained based on the zebrafish survival curves over the entire experimental period, which is defined as the time period from the occurrence of deaths in all groups to the death of the last zebrafish.

Images