Method for evaluating chloride type salt-alkali stress protection effect of fish feed raw material

Abstract

The invention provides a method for evaluating the chloride type salt-alkali stress protection effect of aquatic animal feed raw materials. Comprising the following steps: 1) mixing aquatic animal feed raw materials to be evaluated with basal feed, feeding zebra fish as a test group, feeding zebra fish with basal feed as a control group, respectively observing the control group and the test group, and recording survival conditions of the zebra fish; and (2) carrying out chloride type salt-alkali stress treatment on the control group and the test group by taking seawater crystals with the salinity of 11-14% o as chloride type salt-alkali stress conditions, evaluating the chloride type salt-alkali stress resistance index of the aquatic animal feed raw materials based on the limited average survival time, and measuring the salt-alkali stress resistance effect of the aquatic animal feed raw materials. According to the method, zebra fish is taken as an object, a standard aquatic feed raw material chloride type salt-alkali stress screening model is established, the aquatic anti-chloride type salt-alkali stress feed raw materials are screened, and a direction is provided for development of aquaculture in chloride type salt-alkali water areas and utilization of feed raw materials.

Description

Technical Field

[0001] This invention belongs to the field of aquatic animal feed ingredients, specifically relating to a method for evaluating the protective effect of chloride-type salt-alkali stress on fish feed ingredients. Background Technology

[0002] my country possesses abundant saline-alkali water resources, mainly distributed in the Northwest, North China, Northeast, and coastal tidal flats. Based on the characteristics of salt and alkali ions in these water bodies, they are primarily classified into chloride-type, carbonate-type, and sulfate-type saline-alkali waters. Chloride-type saline-alkali waters are mainly found in my country's coastal areas. Due to their high salinity, high alkalinity, and low oxygen levels, traditional freshwater aquaculture is difficult to conduct in these waters. Saline-alkali water aquaculture is an important direction for expanding aquaculture space in my country. my country's saline-alkali water aquaculture potential is enormous, and exploring feed ingredients that can enhance the chloride-type saline-alkali stress stress of fish has significant application value.

[0003] The core challenge of saline-alkali water aquaculture is the negative impact of high salinity, high alkalinity, and ion imbalance on fish osmotic regulation, metabolism, and immunity. Research in saline-alkali water fisheries mainly focuses on species selection and the elucidation of salt-alkali stress mechanisms. Currently, there is no screening model for chloride-type salt-alkali stress in fish feed ingredients to study the differences in the protective effects of different feed ingredients against chloride-type salt-alkali stress in fish. Summary of the Invention

[0004] The purpose of this invention is to overcome the gaps in existing research and provide a method for evaluating the protective effect of chloride-type saline-alkali stress on aquatic animal feed ingredients.

[0005] The method for evaluating the protective effect of chloride-type salt-alkali stress in aquatic animal feed ingredients provided by the present invention includes the following steps: 1) The feed ingredients of the aquatic animals to be evaluated were mixed with the basal feed and fed to zebrafish as the experimental group, while the zebrafish were fed the basal feed as the control group. The survival status of the control group and the experimental group was observed and recorded. 2) Using sea salt crystals with a salinity of 11‰-14‰ as the chloride-type salt-alkali stress condition, the control group and the experimental group were subjected to chloride-type salt-alkali stress treatment. The chloride-type salt-alkali stress resistance index of the aquatic animal feed ingredients was evaluated based on the limited mean survival time, and the chloride-type salt-alkali stress resistance effect of the aquatic animal feed ingredients was measured.

[0006] In step 2) of the above method, the ratio of the mean survival time of the chloride-induced salt-alkali stress survival curves of the control group and the experimental group is calculated according to the following formula to obtain the chloride-induced salt-alkali stress resistance index of the aquatic animal feed raw material. The calculation formula is: Chloride-resistant salt-alkali stress index of aquatic animal feed ingredients = B / A Where A represents the control group's chloride-type saline-alkali stress survival curve limiting the mean survival time. B represents the limiting mean survival time of the chloride-type saline-alkali stress survival curve for the experimental group.

[0007] The larger the index value, the better the resistance to chloride-induced salt and alkali stress of the aquatic animal feed ingredients.

[0008] The chloride-resistant salt-alkali stress index of fishmeal ranges from 0.88 to 1.24. The chloride-resistant salt-alkali stress index of soybean meal ranges from 0.97 to 1.66.

[0009] In the above method, the aquatic animal can be a fish; The aquatic animal feed ingredients to be evaluated were mixed with the basal feed at a mass ratio of 4-1:6-9. The feeding period (trial period) can be 10-20 days; After the experiment ended, the zebrafish in each group were fasted for 24 hours and then weighed. After weighing for 24 hours, each group of zebrafish was transferred to a sea salt solution (which had been aerated in advance) for chloride-type saline-alkali stress treatment. The salinity of the sea salt in the sea salt solution can be 11‰-14‰, and the mass concentration can be 11.3g / L-14.4g / L; Change the water every 24 hours during the stress period; Aquaculture water temperature: 26-28℃; dissolved oxygen: >6.5 mg / L; Mortality was recorded in real time during the stress period, and the survival curves and corresponding limited mean survival time of the control group and the experimental group under chloride-type saline-alkali stress were statistically analyzed.

[0010] 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 [Lu Beier, Miao Pengcheng, Ma Rongji, et al. Introduction to the nonproportional risk model based on limiting mean survival time and its SAS implementation [J]. Chinese Health Statistics, 2024, 41(05):757-761].

[0011] Limited mean survival (MMS) is an important indicator in survival analysis. It considers the average survival time within a specific time limit and can more comprehensively reflect the characteristics of the survival curve. Calculating the difference in survival curves based on MMS allows for a more flexible consideration of survival at different time points, especially when survival curves intersect or the disproportionate risk assumption is not valid, providing more accurate comparison results [Huang Xinghui, Chen Jinbao, Yang Zijing, et al. Research on nonparametric statistical inference method based on area between two survival curves [J]. Chinese Journal of Health Statistics, 2019, 36(01):8-12.].

[0012] The relative effect can be measured by the ratio of the restricted mean survival time of the experimental group to that of the control group [Lu Beier, Miao Pengcheng, Ma Rongji, et al. Introduction to the non-proportional risk model based on restricted mean survival time and its SAS implementation [J]. Chinese Journal of Health Statistics, 2024, 41(05):757-761, Dehbi HM, Royston P, Hackshaw A. Life expectancy difference and life expectancy ratio: two measures of treatment effects in randomised trials with non-proportional hazards. BMJ. 2017 May 25;357:j2250.doi: 10.1136 / bmj.j2250. PMID: 28546261; PMCID: PMC5444092].

[0013] This experiment, based on a small sample size and short-cycle survival curve comparison, calculated the restricted mean survival time (RMS) of different feed ingredients throughout the entire lifespan. The ratio of the restricted mean survival time of the experimental group to that of the control group was used to measure the difference in efficacy between the two groups, and a qualitative description was provided. This invention uses zebrafish as the target species and establishes a standard screening model for chloride-induced salinity stress in aquatic feed ingredients. It screens aquatic feed ingredients resistant to chloride-induced salinity stress, providing direction for the development of aquaculture in chloride-induced salinity waters and the utilization of feed ingredients. Attached Figure Description

[0014] Figure 1 This is for screening the concentration of chloride-type saline-alkali stress sea salt in zebrafish in Example 1 of the present invention.

[0015] Figure 2 The survival curves of zebrafish under chloride-type saline-alkali stress after being fed different fishmeal and soybean meal in Example 2 of the present invention are shown.

[0016] Figure 3This is to illustrate the repeatability of the chloride-resistant salt-alkali stress index assessment of different feed ingredients based on limited mean survival time in Example 4 of the present invention. Detailed Implementation

[0017] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0018] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0019] The zebrafish used in this study were provided by the Aquatic Microbiology and Feed Innovation Team of the Feed Research Institute of the Chinese Academy of Agricultural Sciences, and all of them were AB strains.

[0020] The basic feed ingredients, proportions, and nutritional levels for zebrafish are shown in Table 1.

[0021] Table 1. Basic Feed Ingredients and Proportions

[0022] Example 1: Screening of chloride-induced saline-alkali stress concentrations in zebrafish One hundred and forty healthy zebrafish juveniles of uniform size, weighing (40±5) mg, were randomly selected for the experiment 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 reared zebrafish were randomly divided into seven groups of 20 each. The seven groups were placed in seawater solutions with salinities of 10‰, 11‰, 12‰, 13‰, 14‰, 15‰, and 16‰, respectively, to screen for chloride-type salinity stress. The solution was changed every 24 hours, and the stress was maintained with 12 hours of light and 12 hours of darkness per day. The fish were fasted during the experiment, and mortality was recorded every 2 hours to plot survival curves.

[0023] Figure 1 Screening for salinity levels in zebrafish to detect acute chloride-induced salinity stress.

[0024] The results showed that a salinity of 11‰-14‰ was the effective salinity for chloride-type salt-alkali stress.

[0025] Example 2: Survival curves of different feed ingredients under chloride-type salt-alkali stress differed in zebrafish. Feed ingredients (using fishmeal and soybean meal as examples) were pulverized and passed through a 60-mesh sieve. Then, the feed ingredients were mixed with the basal feed (Table 1) at a weight ratio of 3:7 and pelleted to prepare experimental feeds, including a basal feed control group, a fishmeal treatment group (two types of fishmeal), and a soybean meal treatment group (six types of soybean meal). Ninety zebrafish with an initial body weight of 40±5 mg were randomly divided into 9 groups of 10 fish each. The control group was fed the basal feed, while the treatment groups were the fishmeal feed group and the soybean meal feed group, respectively. The experiment lasted for 14 days, and the daily feeding amount was recorded.

[0026] The fishmeal, soybean meal, and wheat used in this experiment were all sourced from major domestic aquatic feed companies. Their nutritional components are shown in Table 2 below. Table 2 Nutritional composition of fish meal, soybean meal, and wheat

[0027] After the experiment ended, the zebrafish in each group were fasted for 24 hours and then weighed.

[0028] After weighing for 24 hours, each group of zebrafish was transferred to a pre-aerated 13‰ salinity sea salt solution for chloride-induced salinity stress. The water was changed every 24 hours. Throughout the stress experiment, dead fish were promptly removed, and the culture water temperature was maintained at 27.5℃ with dissolved oxygen >6.5 mg / L. Mortality rates were recorded in real-time during the stress period to improve technical accuracy.

[0029] Figure 2 Survival curves of zebrafish under chloride-type saline-alkali stress fed with different feed ingredients.

[0030] Example 3: Assessment of Chloride-Resistant Salt-Alkali Stress 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 [Lu Beier, Miao Pengcheng, Ma Rongji, et al. Introduction to the nonproportional risk model based on limiting mean survival time and its SAS implementation [J]. Chinese Health Statistics, 2024, 41(05):757-761].

[0031] Limited mean survival (MMS) is an important indicator in survival analysis. It considers the average survival time within a specific time limit and can more comprehensively reflect the characteristics of the survival curve. Calculating the difference in survival curves based on MMS allows for a more flexible consideration of survival at different time points, especially when survival curves intersect or the disproportionate risk assumption is not valid, providing more accurate comparison results [Huang Xinghui, Chen Jinbao, Yang Zijing, et al. Research on nonparametric statistical inference method based on area between two survival curves [J]. Chinese Journal of Health Statistics, 2019, 36(01):8-12.].

[0032] The relative effect can be measured by the ratio of the restricted mean survival time of the experimental group to that of the control group [Lu Beier, Miao Pengcheng, Ma Rongji, et al. Introduction to the non-proportional risk model based on restricted mean survival time and its SAS implementation [J]. Chinese Journal of Health Statistics, 2024, 41(05):757-761, Dehbi HM, Royston P, Hackshaw A. Life expectancy difference and life expectancy ratio: two measures of treatment effects in randomised trials with non-proportional hazards. BMJ. 2017 May 25;357:j2250.doi: 10.1136 / bmj.j2250. PMID: 28546261; PMCID: PMC5444092].

[0033] This experiment, based on a small sample and short-cycle survival curve comparison, calculated the restricted mean survival time of different feed ingredients throughout the entire lifespan, and used the ratio of the restricted mean survival time of the experimental group to the control group as a measure of the difference in effect between the two groups, and provided a qualitative description of it.

[0034] In this experiment, the mean survival time (MST) of the chloride-type saline-alkali stress survival curve in the control group was defined as A, and the mean survival time (MST) of the chloride-type saline-alkali stress survival curve in the experimental group was defined as B. The ratio B / A was used as the chloride-type saline-alkali stress resistance index for feed ingredients. The calculation formula is: The resistance index of a certain raw material to chloride-type salt and alkali stress is B / A, where the larger the index value, the better the resistance of the raw material to chloride-type salt and alkali stress.

[0035] Survival analysis was conducted on two types of raw materials: fishmeal and soybean meal. The average survival time and chloride-resistant salt-alkali stress index were calculated using the formulations in Table 1 as controls. The chloride-resistant salt-alkali stress index of fishmeal ranged from 0.88 to 1.24, with a dispersion of 19.47% among different fishmeals. The chloride-resistant salt-alkali stress index of soybean meal ranged from 0.97 to 1.66, with a dispersion of 18.08% among different soybean meal formulations (see Table 3).

[0036] Table 3 Chloride-resistant salt-alkali stress index of different feed ingredients

[0037] The dispersion represents the difference in stress resistance among the same feed ingredients, with fishmeal showing a difference of 19.47% and soybean meal showing a difference of 18.08%.

[0038] Example 4: Stability Verification of Feed Ingredients' Chloride-Resistant Salt-Alkali Stress Index Based on Limiting Mean Survival Time Based on Table 1, different batches of control feed were prepared to verify the stability of the chloride-resistant salt-alkali stress index results among different batches of the same feed.

[0039] Figure 3 To assess the repeatability of chloride-resistant salt-alkali stress indexes of different feed ingredients based on limited mean survival time.

[0040] Table 4. Dispersion of chloride-resistant salt-alkali stress index for different feed ingredients

[0041] Based on the assessment of chloride-resistant salt-alkali stress index of different feed ingredients by limiting mean survival time, the results showed that the chloride-resistant salt-alkali stress index of fishmeal C was 5.20%; the chloride-resistant salt-alkali stress index of wheat A was 8.71%; and the chloride-resistant salt-alkali stress index of soybean meal G was 7.03% (see Table 4).

[0042] In this invention, zebrafish were used as a model organism. An acute stress experiment determined that a salinity of 11‰-14‰ for sea salt crystals was the effective salinity for chloride-type salinity stress. Next, using the selected effective salinity as the stress condition, different feed ingredients were mixed with a basal diet and fed to zebrafish. Their survival was observed and recorded, and survival curves were plotted. Finally, the resistance index of the feed ingredients to chloride-type salinity stress was evaluated based on the restricted mean survival time. The ratio of the restricted mean survival time of the experimental group to the control group was used to measure the salt-alkali stress resistance effect of the feed ingredients.

[0043] The method of this invention has the advantages of simple operation, accurate results, and good repeatability, providing an effective technical means for the development of aquaculture in chloride-type saline-alkali waters and the utilization of feed raw materials.

[0044] Furthermore, this invention further verifies the stability of the chloride-resistant salt-alkali stress index of feed ingredients based on limited mean survival time. By preparing different batches of control feed, the stability of the chloride-resistant salt-alkali stress index results among different batches of the same feed was verified, and the results show that the method has high stability and reliability.

[0045] The embodiments of this invention use fishmeal and soybean meal as examples for detailed description, but are not limited to these two feed ingredients. In fact, this method can be applied to the screening and evaluation of the anti-chloride-type saline-alkali stress effects of various fish feed ingredients. Through screening and evaluation using this method, higher-quality feed ingredients can be provided for aquaculture in chloride-type saline-alkali waters, improving the fish's resistance to saline-alkali stress and promoting the healthy development of the aquaculture industry.

[0046] In summary, this invention provides a method for evaluating the protective effect of chloride-type saline-alkali stress on fish feed ingredients. This method has broad application prospects and significant practical value. The implementation of this method can provide new ideas and technical means for the development of aquaculture in chloride-type saline-alkali waters and the utilization of feed ingredients, thus promoting the sustainable development of my country's aquaculture industry.

[0047] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. A method for evaluating the protective effect of chloride-type salt-alkali stress on aquatic animal feed ingredients, comprising the following steps: 1) The feed ingredients of the aquatic animals to be evaluated were mixed with the basal feed and fed to zebrafish as the experimental group, while the zebrafish were fed the basal feed as the control group. The survival status of the control group and the experimental group was observed and recorded. 2) Using sea salt crystals with a salinity of 11‰-14‰ as the chloride-type salt-alkali stress condition, the control group and the experimental group were subjected to chloride-type salt-alkali stress treatment. The chloride-type salt-alkali stress resistance index of the aquatic animal feed ingredients was evaluated based on the limited mean survival time, and the chloride-type salt-alkali stress resistance effect of the aquatic animal feed ingredients was measured.

2. The method according to claim 1, characterized in that, The ratio of the chloride-induced salt-alkali stress survival curve limiting mean survival time between the control group and the experimental group was calculated using the following formula to obtain the chloride-induced salt-alkali stress resistance index of the aquatic animal feed ingredient. The calculation formula is: Chloride-resistant salt-alkali stress index of aquatic animal feed ingredients = B / A Where A represents the control group's chloride-type saline-alkali stress survival curve limiting the mean survival time. B represents the limiting mean survival time of the chloride-type saline-alkali stress survival curve for the experimental group.

3. The method according to claim 2, characterized in that, The higher the index value, the better the resistance of the aquatic animal feed ingredients to chloride-induced salt and alkali stress.

4. The method according to claim 1, characterized in that, The aquatic animals mentioned are fish; The aquatic animal feed ingredients to be evaluated were mixed with the basal feed at a mass ratio of 4-1:6-9. The feeding period (trial period) is 10-20 days.

5. The method according to claim 4, characterized in that, After the experiment ended, the zebrafish in each group were fasted for 24 hours and then weighed. After weighing for 24 hours, the zebrafish in each group were transferred to sea salt crystal solution for chloride-type saline-alkali stress treatment. Mortality was recorded in real time during the stress period, and the survival curves and corresponding limited mean survival time of the control group and the experimental group under chloride-type saline-alkali stress were statistically analyzed.

6. The method according to claim 5, characterized in that, Change the water every 24 hours during the stress period; Aquaculture water temperature 26-28℃, dissolved oxygen > 6.5 mg / L.

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