阅读说明：本技术 一种胁迫条件下鱼饲喂条件的筛选方法和一种高温胁迫下鲤科鱼类的饲喂方法 (Method for screening feeding conditions of fishes under stress condition and method for feeding fishes of Cyprinaceae under high-temperature stress ) 是由 李志华 陈成壮 厉萍 于 2021-08-24 设计创作，主要内容包括：本发明涉及水产养殖技术领域,特别是涉及一种胁迫条件下鱼饲喂条件的筛选方法和一种高温胁迫下鲤科鱼类的饲喂方法。本发明利用鱼类普遍具有补偿生长现象的特点,设计合理的间歇性饲喂模式,并通过测定鲤鱼的生长性能指标、肝脏抗氧化能力指标、血清生化参数指标以及肠道消化酶指标,结合整合生物标志物(IBR)指数值,选择其中具有显著性差异的指标作为评价鲤鱼耐高温性能的技术指标,从而确定了高温胁迫下对鲤鱼最佳的饲喂方法,本发明提供的方法可以在高温胁迫时,可以降低饵料的消耗量,降低养殖成本。(The invention relates to the technical field of aquaculture, in particular to a screening method of fish feeding conditions under stress conditions and a feeding method of cyprinid fishes under high-temperature stress. The optimal feeding method for the carps under high-temperature stress is determined by utilizing the characteristic that fishes generally have the effect of compensating growth phenomena, designing a reasonable intermittent feeding mode, measuring the growth performance index, the liver oxidation resistance index, the serum biochemical parameter index and the intestinal digestive enzyme index of the carps, combining and integrating the index values of biological markers (IBR), and selecting the index with significant difference as the technical index for evaluating the high-temperature resistance of the carps.)

1. A screening method of fish feeding conditions under stress conditions is characterized by comprising the following steps:

randomly dividing the fishes with the same specification into a plurality of treatment groups, feeding each group according to different feeding conditions, and culturing for 30-50 days under the same stress condition;

after the culture is finished, randomly taking 3-4 fishes from each group respectively to measure the liver oxidation capacity, the serum biochemical parameters and the intestinal digestive enzyme indexes, and calculating the IBR index;

s ═ Z + | Min |, formula iv;

y ═ X-m)/s, formula v;

a in formula I and formula II_iIs Si and S_i+1And the area of the graph formed by connecting the radii of the star-shaped graph; n in formula I and formula III is the number of measured indexes;

s in formula II and formula III_iAnd S_i+1Two successive scores representing respectively different indices of the clockwise direction of the star plot; wherein S_iAnd S_i+1Calculating according to a formula IV;

s in formula iv is the score for each index and Z is the value of each index after treatment according to formula v, wherein Z is Y when the reaction of the index is activated; when the reaction of the index is inhibited, Z ═ Y; min is the minimum of all measured Y values for each index;

x in formula V is the average of the randomly sampled measured values for each index in each group, where m is the overall average of all treatment groups for each index; s is the standard deviation of all treatment groups for each index;

and judging according to the calculation result, and selecting the feeding condition with the minimum IBR index as the stress-resistant feeding condition.

2. The screening method according to claim 1, wherein the liver antioxidant capacity index includes total superoxide dismutase activity, catalase activity, glutathione peroxidase activity, and malondialdehyde content;

the serum biochemical parameter indicators include glucose level, alanine aminotransferase activity, alkaline phosphatase activity, aspartate aminotransferase activity, total cholesterol content, triglyceride content, and total protein content;

the intestinal digestive enzyme activity index comprises trypsin activity, lipase activity and amylase activity.

3. The screening method according to claim 1, wherein the stress conditions include high temperature stress; the temperature of the high-temperature stress is 28-35 ℃.

4. The screening method according to claim 1, wherein the feeding conditions include a daily continuous feeding group, a one-day interval feeding group, a two-day interval feeding group and a four-day interval feeding group.

5. A feeding method of cyprinid fishes under high temperature stress is characterized by comprising the following steps: feeding the feed at intervals of two days under the high-temperature stress; the temperature of the high-temperature stress is 28-35 ℃.

6. The screening method according to claim 5, wherein the weight ratio of the feed fed by satiety to carp is (3-5): 100.

7. Screening method according to claim 5, wherein the method of satiating feeding comprises two feeds.

8. Screening method according to claim 5 or 7, wherein the time of satiety feeding comprises: 7 am to 9 am and 1 pm to 3 pm.

9. The screening method according to claim 5, wherein the carp family fish includes carp.

Technical Field

The invention relates to the technical field of aquaculture, in particular to a screening method of fish feeding conditions under stress conditions and a feeding method of cyprinid fishes under high-temperature stress.

Background

Carp, named Cyprinus carpio, belonging to Cyprinus carpio, belongs to Cyprinus carpio. In natural environment, carps are influenced by external conditions such as climatic environment, severely changed temperature, deficient food supply and the like, wherein the water temperature is one of factors which have great influence on fish development. The high temperature can promote the stress reaction of the carp and induce the immune response, so that the sensitivity to pathogenic bacteria is enhanced, and the growth and development of the carp are influenced. In order to improve the utilization rate of feed, reduce the cost of feed and labor and achieve better water quality conditions, a scientific feeding method is necessary to be adopted during high-temperature stress, so that the consumption of feed is reduced, and the breeding cost is reduced.

Disclosure of Invention

In order to solve the above problems, the present invention provides a method of screening feeding conditions for fishes under stress and a method of feeding fishes of the family Cyprinaceae under high temperature stress. The method for screening the feeding conditions of the carps under the stress condition can determine the optimal feeding method for the carps under the high-temperature stress, and the feeding conditions screened by the method can reduce the bait consumption and the breeding cost under the high-temperature stress.

In order to achieve the above purpose, the invention provides the following technical scheme:

the invention provides a screening method of fish feeding conditions under stress conditions, which comprises the following steps:

randomly dividing the fishes with the same specification into a plurality of treatment groups, feeding each group according to different feeding conditions, and culturing for 30-50 days under the same stress condition;

after the culture is finished, randomly taking 3-4 fishes from each group respectively to measure the liver oxidation capacity, the serum biochemical parameters and the intestinal digestive enzyme indexes, and calculating the IBR index;

S_n+1＝S₁formula III;

s ═ Z + | Min |, formula iv;

y ═ X-m)/s, formula v;

a in formula I and formula II_iIs Si and S_i+1And the area of the graph formed by connecting the radii of the star-shaped graph; n in formula I and formula III is the number of measured indexes;

s in formula II and formula III_iAnd S_i+1Two successive scores representing respectively different indices of the clockwise direction of the star plot; wherein S_iAnd S_i+1Calculating according to a formula IV;

s in formula iv is the score for each index and Z is the value of each index after treatment according to formula v, wherein Z is Y when the reaction of the index is activated; when the reaction of the index is inhibited, Z ═ Y; min is the minimum of all measured Y values for each index;

x in formula V is the average of the randomly sampled measured values for each index in each group, where m is the overall average of all treatment groups for each index; s is the standard deviation of all treatment groups for each index;

and judging according to the calculation result, and selecting the feeding condition with the minimum IBR index as the stress-resistant feeding condition.

Preferably, the liver antioxidant capacity index comprises total superoxide dismutase activity, catalase activity, glutathione peroxidase activity and malondialdehyde content;

the serum biochemical parameter indicators include glucose level, alanine aminotransferase activity, alkaline phosphatase activity, aspartate aminotransferase activity, total cholesterol content, triglyceride content, and total protein content;

the intestinal digestive enzyme activity index comprises trypsin activity, lipase activity and amylase activity.

Preferably, the stress conditions include high temperature stress; the temperature of the high-temperature stress is 28-35 ℃.

Preferably, the feeding conditions include a daily continuous feeding group, a one-day interval feeding group, a two-day interval feeding group and a four-day interval feeding group.

The invention also provides a feeding method of the cyprinid fishes under high temperature stress, which comprises the following steps: feeding the feed at intervals of two days under the high-temperature stress; the temperature of the high-temperature stress is 28-35 ℃.

Preferably, the weight ratio of the feed for the satiation feeding to the cyprinid fishes is (3-5): 100.

Preferably, the method of satiating feeding comprises two feedings.

Preferably, the time of satiety feeding comprises: 7 am to 9 am and 1 pm to 3 pm.

Preferably, the carp family fish includes carp.

Has the advantages that:

the invention provides a method for screening fish feeding conditions under stress conditions. The optimal feeding method for the carps under high-temperature stress is determined by utilizing the characteristic that fishes generally have the effect of compensating growth phenomena, designing a reasonable intermittent feeding mode, measuring the growth performance index, the liver oxidation resistance index, the serum biochemical parameter index and the intestinal digestive enzyme index of the carps, combining and integrating the index values of biological markers (IBR), and selecting the index with significant difference as the technical index for evaluating the high-temperature resistance of the carps.

Drawings

FIG. 1 shows the response of different feeding methods to the integrated biomarkers in carp under high temperature stress; wherein A is an integrated biomarker radar chart and B is an IBR index value.

Detailed Description

The invention provides a screening method of fish feeding conditions under stress conditions, which comprises the following steps:

randomly dividing the fishes with the same specification into a plurality of treatment groups, feeding each group according to different feeding conditions, and culturing for 30-50 days under the same stress condition;

after the culture is finished, randomly taking 3-4 fishes from each group respectively to measure the liver oxidation capacity, the serum biochemical parameters and the intestinal digestive enzyme indexes, and calculating the IBR index;

S_n+1＝S₁formula III;

s ═ Z + | Min |, formula iv;

y ═ X-m)/s, formula v;

a in formula I and formula II_iIs Si and S_i+1And the area of the graph formed by connecting the radii of the star-shaped graph; n in formula I and formula III is the number of measured indexes;

s in formula II and formula III_iAnd S_i+1Two successive scores representing respectively different indices of the clockwise direction of the star plot; wherein S_iAnd S_i+1Calculating according to a formula IV;

s in formula iv is the score for each index and Z is the value of each index after treatment according to formula v, wherein Z is Y when the reaction of the index is activated; when the reaction of the index is inhibited, Z ═ Y; min is the minimum of all measured Y values for each index;

x in formula V is the average of the randomly sampled measured values for each index in each group, where m is the overall average of all treatment groups for each index; s is the standard deviation of all treatment groups for each index;

and judging according to the calculation result, and selecting the feeding condition with the minimum IBR index as the stress-resistant feeding condition.

In the present invention, the liver antioxidant capacity index preferably includes total superoxide dismutase activity, catalase activity, glutathione peroxidase activity, and malondialdehyde content;

the serum biochemical parameter indicators preferably include glucose level, alanine aminotransferase activity, alkaline phosphatase activity, aspartate aminotransferase activity, total cholesterol content, triglyceride content, and total protein content;

the intestinal digestive enzyme activity index preferably comprises trypsin activity, lipase activity and amylase activity.

In the present invention, the stress conditions preferably include high temperature stress; the high-temperature stress temperature is 28-35 ℃, and preferably 29-31 ℃.

In the present invention, the feeding conditions preferably include a daily continuous feeding group, a one-day interval feeding group, a two-day interval feeding group, and a four-day interval feeding group; the feeding conditions are selected to eliminate the interference of other variables, and preferably one variable is determined for screening to determine the optimal feeding conditions.

The optimal feeding method for the carps under high-temperature stress is determined by utilizing the characteristic that the fishes generally have the effect of compensating the growth phenomenon, designing a reasonable intermittent feeding mode, measuring the growth performance index, the liver oxidation resistance index, the serum biochemical parameter index and the intestinal digestive enzyme index of the carps, combining the Integrated Biomarker (IBR) index value and selecting the index with the significant difference as the technical index for evaluating the high-temperature resistance of the carps.

The invention provides a feeding method of cyprinid fishes under high-temperature stress, which comprises the following steps: feeding the feed every two days under high temperature stress; the temperature of the high-temperature stress is 28-35 ℃.

In the invention, the high-temperature stress temperature is preferably 29-31 ℃, and the cyprinid fishes preferably comprise carps; the weight ratio of the feed fed by the feeding satiation to the cyprinid fishes is preferably (3-5): 100; the feed comprises a Tongwei 103 pond fish culture compound feed of Tongwei group limited company; the method of satiety feeding preferably comprises two feeds; the time of the satiation feeding preferably comprises: 9 am to 11 am and 1 pm to 3 pm.

The method provided by the invention excites the compensating growth potential of the fish by implementing proper intermittent starvation stress, the ingestion speed and the bait utilization efficiency are both improved to a certain extent, the growth speed of the fish body is accelerated, and the technical support is provided for feeding the carp under the condition of continuous high temperature in summer.

To further illustrate the present invention, the screening method of feeding fish under stress and the feeding method of carp under high temperature stress provided by the present invention will be described in detail with reference to the following examples, which should not be construed as limiting the scope of the present invention.

Example 1

A screening process of a feeding method of cyprinid fishes under high temperature stress comprises the following steps:

(1) temporarily breeding the carps: selecting 120 carps with the specification weight of 25.30 +/-1.27 g, randomly dividing the carps into four groups, namely continuously feeding commercial baits every day as a control group (T0), feeding the commercial baits every other day (T1), feeding the commercial baits every other day (T2) and feeding the commercial baits every other day (T3), wherein the commercial baits are compound baits for culturing the carps in Tongwei 103 ponds of Tongwei group Limited company, 30 carps are arranged in each group, three carps are arranged in parallel in each group, 10 carps are arranged in parallel in each group, the carps are transferred to 12 single plastic circulating water aquariums with the volume of 60cm multiplied by 60cm by 120L, and a circulating water culture system is adopted for feeding; in the temporary culture period of two days, the circulating water amount of each tank is 1.0L/min, natural illumination is performed, the water temperature is 25.00 +/-1.00 ℃, and the dissolved oxygen is more than or equal to 5.5mg L^-1The pH value is 7.5 +/-0.2; the temperature condition is controlled by the heating circulating water of the electric heating rod and the room temperature of the double-system air conditioner;

(2) and (3) test treatment: during the high-temperature stress test period, the water temperature is raised to 30 ℃ by an electric heating rod, other water quality conditions are unchanged, and the test period is 40 days; adopting a twice-satiation feeding mode, wherein the weight ratio of the feed for satiation feeding to the carp is 4: 100; the time of the satiation feeding is 8 am and 2 pm, and the residual bait is collected after half an hour of feeding;

(3) collecting a sample: after the test is finished, stopping feeding 24 hours before sample collection, and counting and weighing the carps in each water tank; at the time of sampling, carp was anesthetized with a fishery anesthetic, and the skin of the fish was sterilized with a tampon containing 75 wt.% alcohol; randomly taking 3 carps in each group in parallel for sampling operation; collecting blood sample of Cyprinus Carpio by puncturing tail vein with 2mL syringe, storing in 1.5mL centrifuge tube, centrifuging at 4 deg.C at 7000rpm for 10 min, collecting serum and storing in-80 deg.C refrigerator until biochemical analysis test; dissecting the abdominal cavity of fish, collecting liver, quickly freezing with liquid nitrogen, preserving in a refrigerator at-80 deg.C, and analyzing antioxidant ability parameters; taking the carp midgut, taking out a 2 cm-section of the carp midgut, putting the section into liquid nitrogen, and then putting the section into a refrigerator at the temperature of 80 ℃ below zero for subsequent digestive enzyme activity analysis;

(4) sample treatment: thawing carp liver and intestinal tissue at 4 deg.C, homogenizing with 0.9 wt.% sterile normal saline (tissue weight, g): v (normal saline volume, mL) at a ratio of 10%; centrifuging at 4 deg.C and 3500rpm for 10 min, and collecting supernatant for measuring physicochemical indexes of carp liver and intestinal tract;

(5) the technical indexes are as follows: the physical and chemical indexes of the carp comprise a growth performance index, a liver oxidation resistance index, a serum biochemical parameter index and an intestinal digestive enzyme index; growth performance indicators are weight gain rate (WG), specific weight gain rate (SGR), feed Factor (FCR) and Feed Rate (FR); the antioxidant capacity index includes total superoxide dismutase (T-SOD) activity, Catalase (CAT) activity, glutathione peroxidase (GSH-PX) activity and Malondialdehyde (MDA) content; the serum biochemical parameter index is glucose (Glu) level, alanine amino transfer (ALT) activity, alkaline phosphatase (ALP) activity, aspartate Aminotransferase (AST) activity, Total Cholesterol (TC) content, Triglyceride (TG) content and Total Protein (TP) content; the intestinal digestive enzyme activity index is Trypsin (TRY) activity, Lipase (LPS) activity and Amylase (AMS) activity;

the growth performance index calculation formula is as follows:

weight gain WG (%) - (end weight-initial weight)/initial weight × 100%

Specific growth rate SGR (%. day)^-1) Becoming [ ln (end body weight) -ln (initial body weight)]Day/dayNumber x 100%

Bait coefficient FCR (%) -. total feed intake/(end weight-initial weight) × 100%

Food intake rate FR (%) — total food intake/(days × (end body weight + initial body weight)/2) × 100%

Measuring antioxidant capacity index (T-SOD activity, CAT activity, GSH-PX activity and MDA content), serum biochemical parameters (Glu content, ALT activity, ALP activity, AST activity, TC content, TG content and TP content) and intestinal tract digestive enzyme activity index (TRY activity, LPS activity and AMS activity) according to the specification of kit for measuring Nanjing institute of bioengineering;

the results of the growth performance calculations are shown in table 1; the indexes of the antioxidant capacity of the liver are shown in a result table 2; the serum biochemical parameter index results are shown in table 3; the intestinal tract digestive enzyme activity indexes are shown in a result table 4;

TABLE 1 Effect of different feeding methods on carp growth Performance under high temperature stress

Note: multiple comparison by Duncan's method, wherein data are marked with different letters to indicate that significant difference exists among groups (P < 0.05);

TABLE 2 Effect of different feeding methods on carp liver antioxidant capacity under high temperature stress

Note: multiple comparison by Duncan's method, wherein data are marked with different letters to indicate that significant difference exists among groups (P < 0.05);

TABLE 3 influence of different feeding methods on biochemical parameters of carp serum under high temperature stress

Note: multiple comparison by Duncan's method, wherein data are marked with different letters to indicate that significant difference exists among groups (P < 0.05);

TABLE 4 influence of different feeding methods on carp intestinal digestive enzyme activity under high temperature stress

Note: multiple comparison by Duncan's method, wherein data are marked with different letters to indicate that significant difference exists among groups (P < 0.05);

(6) integrated Biomarker response analysis (integrated Biomarker Responses, IBR): combining the liver oxidation ability index, the serum biochemical parameter index and the intestinal digestive enzyme index as biomarkers for comprehensive analysis, and calculating an IBR index;

the calculation step of the IBR index adopts the following method: calculating the average value X of three repeated samples of each treatment group;

data normalization: processing X to obtain Y, where m is the overall average of all treatment groups per biomarker; s is the standard deviation of all treatment groups for each biomarker; when the response of the biomarker is activated, Z ═ Y; when the response of the biomarker is inhibited, Z ═ Y; finding out the minimum value of Y values of all treatment groups of each biological index, and marking as Min; processing the Min absolute value, and adding the Min absolute value and Z to obtain a score S, wherein S is Z + | Min | and S is more than or equal to 0;

marking the processed data of each biological index in a star chart as a score S of one biological index of each processing group; s_iAnd S_i+1Respectively representing two successive scores in the clockwise direction of the star chart, n being the number of radii, i.e. measuredThe number of physical indexes; a. the_iIs Si and S_i+1And the area of the graph formed by connecting the radii of the star chart:

S_n+1＝S₁formula III;

note: s_iAnd S_i+1When both are 0, beta cannot be calculated, A_iSet to 0;

(7) data processing: performing data analysis by IBM SPSS 25.0 statistical software, wherein the physicochemical index data of the carp is represented by Mean value plus or minus standard error (Mean plus or minus SE); analyzing and processing the obtained data by One-way ANOVA analysis and Duncan's method multiple comparison; the judgment rule that the technical indexes are different remarkably is that P <0.05 represents a remarkable difference.

As can be seen from table 1, the weight gain rate, specific weight gain rate and diet coefficient were significantly higher in the two-day-interval feeding group (T2) than in the other groups (P <0.05), and the food intake rate was significantly higher in the control group (T0) and the one-day-interval group (T1) than in the other groups (P < 0.05);

as can be seen from Table 2, the T-SOD activity was high in the two-day interval feeding group (T2); no significant difference in CAT activity among groups (P > 0.05); low GSH-PX activity in the two-day-spaced group (T2); the minimum MDA content in the control group (T0);

as can be seen from table 3, the highest GLU content (P <0.05) was found in the two-day interval feeding group (T2); the highest ALT activity in the one-day-interval fed group (T1); the lowest ALP activity and AST activity in the control group (T0); the TC content has no significant difference among the groups (P > 0.05); high TG and TP content in the control group (T0);

as can be seen from Table 4, the highest TRY activity was observed in the two-day-spaced feeding group (T2); the lowest LPS activity in the control group (T0); the highest AMS activity in the one day interval group (T1);

as can be seen from fig. 1, there was the lowest IBR index value in the two-day-spaced feeding group (T2), i.e. the T2 group had the lowest stress index of the environment.

In conclusion, under high temperature stress, carp has the best growth performance and digestive enzyme activity in the feeding group (T2) at two-day intervals; the invention excites the compensating growth potential of fish by implementing intermittent hunger stress, the ingestion speed and the bait utilization efficiency are both improved to a certain extent, the growth speed of the fish body is accelerated, and the scientific understanding of the carp feeding strategy under the condition of continuous high temperature in summer is further deepened.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.