Evaluation method for low-temperature stress protection effect of fish feed raw materials

Abstract

The invention discloses a method for evaluating the low-temperature stress protection effect of fish feed raw materials. The evaluation 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; s2, after fasting, performing acute low-temperature stress on the zebra fish, observing and recording death time until all the fishes die, and recording a survival curve of the zebra fish; s3, calculating the acute low-temperature stress resistance indexes of different feed raw materials through the average survival time, and measuring the protection difference of different raw materials; the temperature of the acute low-temperature stress is 11-12 DEG C. Based on raw material index data, the energy level and digestibility of the feed are optimized in a targeted manner, and the overwintering survival rate is increased. The method can be applied to scenes such as southern cold waves and northern overwintering, and growth inhibition and immunity decline caused by low temperature are relieved. According to the invention, a reproducible model platform is provided for research on a fish low-temperature stress mechanism.

Description

TECHNICAL FIELD

[0001] The present application relates to a method for evaluating the low-temperature stress protection effect of fish feed raw materials, belonging to the field of aquatic animal feed raw materials. BACKGROUND

[0002] Aquaculture environment is affected by nature. Fish are mostly poikilothermic animals and cannot maintain stable body temperature. When the environmental temperature is lower than the suitable range for fish growth, fish are prone to low-temperature stress. In the field of aquaculture in China, winter cold invasion in the south, wintering in the north, and diurnal temperature difference in high-altitude areas, as well as early stocking in spring, all can cause significant temperature changes. In cage culture, excessive water flow can also easily cause large fluctuations in water temperature. These scenarios are prone to cause low-temperature stress in fish.

[0003] Low-temperature stress mainly affects the physiological processes of fish by inducing a decrease in biological enzyme activity and cell membrane fluidity, and low-temperature stress is one of the main reasons affecting the efficiency of aquaculture. In water production and feed formulation, the composition of feed raw materials and the addition of functional ingredients are often adjusted to enhance the ability of fish to resist low-temperature stress and alleviate the negative effects of low-temperature stress on growth, immunity, etc.

[0004] When fish are under low-temperature stress, the metabolic rate decreases, and the energy level in the feed needs to be increased; the digestive enzyme activity in the fish decreases, and high-digestibility feed raw materials need to be selected. At the same time, feed raw materials contain varying degrees of antioxidant components, such as vitamin C, vitamin E, polysaccharides, etc. Therefore, it is of great significance to comprehensively evaluate the protection effect of feed raw materials on fish under low-temperature stress and further customize cold-resistant formulations for fish in the field of aquaculture and feed.

[0005] At present, there is no fish feed raw material low-temperature stress screening model for studying the differences in the low-temperature protection effect of different feed raw materials on fish, resulting in no evaluation standard and use direction for the low-temperature stress resistance level of feed raw materials. Therefore, the present application takes zebrafish as the object and establishes a standard aquaculture feed raw material low-temperature stress screening model, so as to better carry out research on the response of feed raw materials to low-temperature stress in fish and screen aquatic low-temperature stress resistant feed raw materials. SUMMARY

[0006] The purpose of the present application is to overcome the existing gaps in the prior art and provide a method for evaluating the low-temperature stress of fish feed raw materials, for screening and evaluating the role of different feed raw materials in alleviating low-temperature stress in fish; the present application solves the problem of lack of unified evaluation standard in the prior art by constructing a repeatable low-temperature stress model and applying the RMST (restricted mean survival time) index, so that the low-temperature stress resistance of feed raw materials can be quantified and compared, thereby optimizing the formulation of aquatic feed and improving the efficiency of aquaculture.

[0007] The application provides an evaluation method for low-temperature stress protection effect of fish feed raw materials, and comprises the following steps: S1, mixing and granulating the feed raw material to be evaluated and a basic feed to prepare a test feed, and feeding zebra fish; S2, after fasting, subjecting the zebra fish to acute low-temperature stress, observing and recording the death time until all the fish die, and recording the survival curve of the zebra fish; The temperature of the acute low-temperature stress is 11-12 DEG C.

[0008] Preferably, the feed raw material comprises fish meal or soybean meal, which is crushed to pass through a 60-mesh sieve and mixed with the basic feed.

[0009] Preferably, the basic feed comprises casein, gelatin, dextrin, soybean oil, lysine, vitamin C, multi-vitamin, multi-mineral, calcium dihydrogen phosphate, choline, sodium alginate and zeolite powder. The mass ratio of the feed raw material to the basic feed is 1-4:9-6, preferably 3:7.

[0010] In the screening method, the initial weight of the zebra fish is (30-60) ± 5 mg; The feeding time is 10-14 days. The fasting time is 24-48 h.

[0011] In the evaluation method, the conditions of the acute low-temperature stress are as follows: The water body is replaced every 12-24 hours, and an 8-12 hour light / dark cycle is maintained.

[0012] The method further comprises the step of determining an anti-low-temperature stress index according to the survival curve of the zebra fish, so as to evaluate the anti-low-temperature stress protection effect of the feed raw material. The anti-low-temperature stress index = B / A B represents the limited average survival time of the test group, and A represents the limited average survival time of the control group. The limited average survival time is obtained based on the survival curve of the zebra fish in the whole test period, and the whole test period is defined as the time period from the beginning of the death of all groups to the end of the death of the last zebra fish.

[0013] The limited average survival time is an important index in survival analysis, which considers the average survival time within a certain time limit, and can more comprehensively reflect the characteristics of the survival curve. Based on the limited average survival time, the difference of the survival curve can be calculated, which can more flexibly consider the survival situation at different time points, especially when the survival curve exists intersection or the proportional hazards assumption is not established, and more accurate comparison results can be provided.

[0014] The greater the value of the anti-low temperature stress index is, the better the protection effect is.

[0015] The present application first constructs a standardized low temperature stress model by taking zebrafish as a model animal, and solves the problem of lacking an evaluation system for quickly screening feed raw materials with anti-low temperature stress in the field of aquatic products.

[0016] The present application calculates the anti-low temperature stress index based on RMST, converts the effect of raw materials into comparable numerical values (index = B / A); RMST can effectively solve the problem of survival curve crossing or non-proportional risk, and avoid the limitations of traditional methods (such as median survival time); by combining survival curve and index, the performance of raw materials can be clearly distinguished.

[0017] Based on the index data of raw materials, the present application optimizes the energy level and digestibility of feed, and improves the overwintering survival rate. The present application can be applied to scenes such as cold wave in the south and overwintering in the north, and can alleviate the growth inhibition and immune decline caused by low temperature. The present application provides a reproducible model platform for the study of low temperature stress mechanism of fish. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 Example 1 of the present application is the screening of acute low temperature stress temperature of zebrafish; Figure 2 Example 2 of the present application is the low temperature stress survival curve of zebrafish with different feed raw materials; Figure 3 Example 3 of the present application is the survival curve of three times of low temperature stress repetition of the same feed raw material; Figure 4 Example 4 of the present application is the repeatability of the anti-low temperature stress index of different feed raw materials based on the limited average survival time. DETAILED DESCRIPTION

[0019] The following examples facilitate better understanding of the present application, but do not limit the present application. In the following examples, the experimental methods are conventional methods unless otherwise specified. In the following examples, the test materials used are commercially available from conventional biochemical reagent stores unless otherwise specified.

[0020] In the following examples, the basic feed raw material composition, ratio and nutrient level of zebrafish are shown in Table 1: Table 1 Basic feed raw material composition and ratio

[0021] Example 1, screening of acute low temperature stress temperature of zebrafish 80 healthy and uniform zebrafish larvae with a weight of (40±5) mg were randomly selected for the test and temporarily fed with basic feed. After 2 weeks of temporary feeding, all zebrafish were starved for 24 h, and then weighed. After weighing, the temporarily fed zebrafish were randomly divided into 4 groups, 20 zebrafish in each group. The 4 groups were respectively placed in water with a water temperature of 9℃, 10℃, 11℃ and 12℃ to screen the acute low-temperature stress temperature. The water in each system was replaced by 1 / 2 of water with sufficient aeration and the same temperature every day, and the light and darkness were kept at 12 h each day, and the pH was kept at 7.0-7.5. During the test period, the zebrafish were starved, and the death of the zebrafish was counted every 2 h and the survival curve was drawn.

[0022] The zebrafish used in the application were provided by the Feed Research Institute of Chinese Academy of Agricultural Sciences, and all were AB strains.

[0023] Figure 1 The results shown in the table show that under the condition of 9-10℃, the zebrafish low-temperature stress dies rapidly; under the condition of 11℃, the survival period of zebrafish low-temperature stress is controlled at 80-100h; under the condition of 12℃, the survival rate of zebrafish low-temperature stress is 80% at 20h; 11-12℃ can be used as the preferred temperature for acute low-temperature stress of zebrafish.

[0024] Example 2, using zebrafish as a model, the survival curves of different feed raw materials under acute low-temperature stress are different, taking fish meal, soybean meal and wheat as examples The feed raw materials, taking fish meal, soybean meal and wheat as examples, were respectively crushed and passed through a 60-mesh sieve. Then, the feed raw materials were mixed with the basic feed (Table 1) at a ratio of 3:7 to prepare the test feed, which was respectively the basic feed control group, the fish meal treatment group, the soybean meal treatment group and the wheat treatment group. 60 zebrafish with an initial weight of 40±5mg were selected and randomly divided into 4 groups, 3 replicates in each group, and 10 zebrafish in each replicate. Among them, the control group was fed with basic feed, and the treatment groups were respectively fish meal feed group, soybean meal feed group and wheat feed group. The test period was 14 days, and the feeding amount was recorded every day during the test period.

[0025] The fish meal, soybean meal and wheat used in the application are all from domestic major aquatic feed enterprises.

[0026] Table 2 Composition and ratio of different test feeds

[0027] After the test period, the zebrafish in each group were starved for 24 h and then weighed.

[0028] After 24 h of weighing, the zebrafish in each group were respectively transferred to 10℃ low-temperature water which had been aerated in advance for acute low-temperature stress. During the test period, the dead fish were taken out in time, and the water was replaced every 24 h. During the whole stress test period, the water temperature was kept at 10±0.5℃.

[0029] Real-time recording of mortality rates during stress periods improves counting accuracy.

[0030] Figure 2 The results show that zebrafish fed with different fishmeals, soybean meals, and wheat showed significant differences in their low-temperature stress survival curves compared to the control feed.

[0031] Example 3: Assessment of Low Temperature Stress Resistance Index of Feed Ingredients Based on Limited Mean Survival Time 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.

[0032] 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.

[0033] This invention calculates the restricted mean survival time of different feed ingredients throughout their entire lifespan based on small sample and short-cycle survival curve differences. The ratio of the restricted mean survival time of the experimental group to that of the control group is used as a measure of the difference in effect between the two groups, and is qualitatively described.

[0034] In this invention, the statistical period is defined as the time from the occurrence of mortality in all groups to the death of the last zebrafish in all experimental groups. Throughout the experimental period, the mean survival time limited by the low-temperature stress survival curve for the control group is calculated as A, and the mean survival time limited by the low-temperature stress survival curve for the experimental groups is calculated as B. The ratio B / A is used as the low-temperature stress resistance index of the feed ingredients. The calculation formula is: The low-temperature stress index of a certain raw material is calculated as B / A, where a larger index value indicates a better resistance to low-temperature stress.

[0035] Survival analysis was conducted on three raw materials: fishmeal, soybean meal, and wheat. Mean survival time and low-temperature stress resistance index were calculated. The results, including two types of fishmeal, two types of soybean meal, and two types of wheat, are shown in Table 3. Figure 3 As shown in the figure, the mean low-temperature stress resistance index of the two types of fishmeal was 1.18, with a dispersion of 27.46%; the mean low-temperature stress resistance index of the two types of soybean meal was 0.99, with a dispersion of 27.84% among the different types of soybean meal; and the mean low-temperature stress resistance index of the two types of wheat was 0.43, with a dispersion of 88.21%.

[0036] Table 3 Low-temperature stress resistance index of different feed ingredients

[0037] Example 4: Stability Verification of Feed Ingredient Low Temperature Stress Index Assessment Based on Limited Mean Survival Time Different batches of control feed were prepared according to Table 1 to test the stability of the results of the low-temperature stress resistance index between different batches of the same feed, and the results are shown in Table 2. Figure 4 The dispersion degree of the low-temperature stress resistance index of different feed raw materials (dispersion degree = standard deviation / average value) is shown in Table 4.

[0038] Table 4 Dispersion degree of low-temperature stress resistance index of different feed raw materials

[0039] The low-temperature stress resistance index of different feed raw materials was evaluated based on the limited average survival time. The results showed that the dispersion degree of the low-temperature stress resistance index of fish meal C was 3.68%; the dispersion degree of the low-temperature stress resistance index of soybean meal C was 3.91%; the dispersion degree of the low-temperature stress resistance index of wheat C was 3.84%; and the dispersion degree of the low-temperature stress resistance index of wheat D was 2.03%, indicating that the results of the low-temperature stress resistance index of different feed raw materials were good in reproducibility.​

Claims

1. A method for evaluating the low-temperature stress protection effect of a fish feed raw material, comprising the following steps: S1, mixing and granulating a feed raw material to be evaluated with a basic feed to prepare a test feed, and feeding zebrafish; S2, after fasting, subjecting the zebrafish to acute low-temperature stress, observing and recording the death time until all the fish die, and recording the survival curve of the zebrafish; The temperature of the acute low-temperature stress is 11-12℃.

2. The evaluation method according to claim 1, characterized by: The feed raw material includes fish meal or soybean meal, which is ground to pass through a 60-mesh sieve and mixed with the basic feed.

3. The evaluation method according to claim 1 or 2, characterized by: The basic feed includes casein, gelatin, dextrin, soybean oil, lysine, vitamin C, multi-vitamin, multi-mineral, calcium dihydrogen phosphate, choline, sodium alginate and zeolite powder. The mass ratio of the feed raw material to the basic feed is 1-4:9-6.

4. The evaluation method according to any one of claims 1 to 3, characterized by: The initial weight of the zebrafish is (30-60) ±5 mg. The feeding time is 10-14 days. The fasting time is 24-48 h.

5. The evaluation method according to any one of claims 1 to 4, characterized by: The conditions of the acute low-temperature stress are as follows: The water is changed every 12-24 hours, and the light / dark cycle is maintained for 8-12 hours.

6. The evaluation method according to any one of claims 1 to 5, characterized by: The method further comprises a step of determining an anti-low-temperature stress index according to the survival curve of the zebrafish to evaluate the anti-low-temperature stress protection effect of the feed raw material; The anti-low-temperature stress index = B / A B represents the limited average survival time of the test group, and A represents the limited average survival time of the control group. The limited average survival time is obtained based on the survival curve of the zebrafish in the whole test period, which is defined as the time period from the occurrence of death in all groups to the death of the last zebrafish.

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