CN112021473A - Composite additive and feed for reducing eating behavior of takifugu rubripes - Google Patents

Composite additive and feed for reducing eating behavior of takifugu rubripes Download PDF

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CN112021473A
CN112021473A CN202011040974.4A CN202011040974A CN112021473A CN 112021473 A CN112021473 A CN 112021473A CN 202011040974 A CN202011040974 A CN 202011040974A CN 112021473 A CN112021473 A CN 112021473A
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feed
rubripes
compound additive
lysine
methionine
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卫育良
梁萌青
徐后国
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Yellow Sea Fisheries Research Institute Chinese Academy of Fishery Sciences
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Abstract

The invention discloses a compound additive and feed for reducing the eating behavior of fugu rubripes, belongs to the field of aquatic animal nutrition, and aims at the serious eating behavior of the fugu rubripes in the cultivation process. By applying different dosages and combinations of the complex additives (lysine, methionine, cysteine, dimethyl-beta-propitioxetine DMPT and tryptophan) under the typical cultivation conditions of the takifugu rubripes. After the compound additive is added in indoor factory culture in winter and offshore cage culture in summer, the discovery can not only obviously reduce mutual attack, reduce cannibalism behavior, but also improve the survival rate of the compound additive in high-temperature seasons, and increase culture benefits. This shows that the comprehensive nutrition method of the invention has good application effect on reducing the cannibalism behavior in the full-period cultivation process of the takifugu rubripes.

Description

Composite additive and feed for reducing eating behavior of takifugu rubripes
Technical Field
The invention belongs to the field of aquatic animal nutrition, and particularly relates to a compound additive and feed for reducing eating habits of Takifugu rubripes.
Background
The red-fin eastern globefish is one of the main globefish breeding varieties, and is always a traditional delicacy in three countries of China, China and China, due to the specific nutritive value and the flavor of the red-fin eastern globefish. The cultivation of the takifugu rubripes in China starts from the last 80 th century and continues to the present, and is the country with the largest cultivation yield at present, but because wild takifugu rubripes contain tetrodotoxin, China prohibits the takifugu rubripes from entering market for a long time, and the cultivated takifugu rubripes are mainly sold to korea and japan. In 2016, the 'fasting order' of puffer fish has been conditionally released, which is a new development opportunity for the cultivation and consumption of fugu rubripes.
The breeding problem of the takifugu rubripes from the beginning to the present is mutual cannibalism among the takifugu rubripes, and the cannibalism not only exists in a juvenile fish stage, but also often appears in a breeding stage, so that the survival rate of breeding is reduced, and the breeding benefit and the economic income of fishermen are influenced. In the production practice, in order to solve the problem of cannibalism of the takifugu rubripes, measures such as regularly clipping teeth of the takifugu rubripes, reducing the breeding density and the like are generally adopted, but the breeding measures not only increase the labor intensity of people, but also have limited effect on relieving the cannibalism of the takifugu rubripes. Therefore, the cultivation of the fugu rubripes urgently needs to use a nutrition regulation technology to invent a high-efficiency comprehensive nutrition method, so that the eating behavior of the fugu rubripes in cultivation is reduced, and the cultivation yield and benefit are improved.
Disclosure of Invention
The invention aims to provide a compound additive and feed for reducing the eating behavior of fugu rubripes. Aiming at the serious cannibalism behavior in the cultivation process of the fugu rubripes, the invention improves the existing commercial feed by a nutriology regulation and control means, and the compound additive (lysine, methionine, cysteine, dimethyl-beta-propitioxetine DMPT and tryptophan) is added into the feed to reduce the cannibalism behavior of the fugu rubripes under the typical cultivation condition.
The invention is realized according to the following technical scheme:
a compound additive for reducing the eating behavior of fugu rubripes comprises lysine, methionine, cysteine, DMPT and tryptophan.
The composite additive feed for red-fin eastern globefish has the overwintering indoor culture condition of 30-300g weight, the culture water temperature is maintained at 16-18 deg.c, and on the basis of commercial feed, the composite additive is added into the feed, and the mass ratio of the composite additive in the feed to the dry matter of the feed is lysine 4.05%, methionine 1.12%, cysteine 2.32%, DMPT 0.03% and tryptophan 0.86%.
The takifugu rubripes feed added with the compound additive is characterized in that under the condition that takifugu rubripes with the weight of 300-1000g is cultured in a marine net cage in summer, the culture water temperature is 20-28 ℃, and the compound additive is added on the basis of commercial feed, so that the mass ratio of each component of the compound additive in the feed to dry matter of the feed is respectively 3.73% of lysine, 1.05% of methionine, 2.12% of cysteine, 0.03% of DMPT and 0.77% of tryptophan.
Furthermore, lysine, methionine, cysteine and tryptophan are all L-shaped, and the purity is more than 99%; DMPT with purity of more than 98%; "4.05% lysine" means that the content of lysine in the feed is 4.05% of the mass of dry matter of the feed; the contents of methionine, cysteine, DMPT and tryptophan in the feed are expressed in the same manner as those of lysine.
Compared with the prior art, the invention has the beneficial effects that:
(1) the compound additive can obviously improve the content of 5-hydroxytryptamine in the brain of the takifugu rubripes, so that the fish can generate a pleasant mood and mutual attack behaviors are reduced.
(2) The invention obviously improves the palatability of the feed, reduces the cannibalism behavior of the takifugu rubripes under different culture conditions, and improves the survival rate of the fish in high-temperature seasons.
(3) The additive used in the invention has been industrialized, and has strong operability, safety and reliability.
Drawings
FIG. 1 effect of feed lysine levels on the rate of eating by takifugu rubripes;
FIG. 2 effect of feed methionine level on the cannibalism rate of Fugu rubripes;
FIG. 3 effect of feed cysteine levels on the cannibalism rate of Fugu rubripes;
FIG. 4 effect of feed DMPT level on cannibalism rate of Fugu rubripes;
FIG. 5 effect of feed tryptophan level on the cannibalism rate of Fugu rubripes;
FIG. 6 the 5-hydroxytryptamine content in the brain of Fugu rubripes;
FIG. 78 survival at the end of the cultivation experiment;
FIG. 8 shows the 5-hydroxytryptamine content in the brain of Fugu rubripes under indoor industrial cultivation conditions in winter;
FIG. 9 shows mutual cannibalism of Fugu rubripes under indoor industrial cultivation conditions in winter;
FIG. 10 the cultivation survival rate of Fugu rubripes under indoor industrial cultivation conditions in winter;
FIG. 11 shows the 5-hydroxytryptamine content in the brain of Fugu rubripes under the marine cage culture conditions;
FIG. 12 shows mutual cannibalism of Fugu rubripes under marine cage culture conditions;
FIG. 13 survival rate of Fugu rubripes under marine cage culture conditions.
Detailed description of the preferred embodiments
The technical solutions of the present invention are further illustrated by the following specific examples, but the scope of the present invention is not limited in any way by the examples:
example 1 screening of additives for reducing eating behavior of Fugu rubripes and the procedure thereof
(1) Design of experiments
All basic feeds for screening experiments are semi-refined feeds prepared from fish meal, soybean meal, zein, gelatin and mixed amino acids, and gradient lysine, methionine, cysteine, DMPT or tryptophan are added on the basis of the semi-refined feeds to perform gradient screening experiments on the residual eating behaviors of 5 additives. Wherein, the lysine screening experiment only supplements 0.3 percent of crystal methionine to ensure that the methionine content of the feed is 0.89 percent; methionine experiments were supplemented with only 1.5% crystalline lysine, resulting in a lysine level of 3.05%; cysteine, DMPT and tryptophan screening experiments were each supplemented with 0.3% crystalline methionine and 1.5% crystalline lysine, respectively, to achieve feed methionine and lysine levels of approximately 0.89% and 3.05%, respectively. The different levels of screening in the feed for the corresponding additives for each experimental feed are shown in table 1.
TABLE 1 actual content (%, dry matter of feed) of the different additive packages in the feed
Figure BDA0002706643640000041
(2) Experimental fish and breeding management
Before the experiment begins, all the cultured fishes are subjected to tooth shearing treatment in order to ensure the culture survival rate and reduce the over-high death rate caused by the cannibalism. After cutting teeth, temporarily culturing for 2 weeks, starting formal culturing experiment, selecting Fugu rubripes with weight of about 15g for 5 groups of initial fish, placing into culturing barrel (square, volume of 0.7m × 0.7m × 0.4m), placing 30 fish in each barrel, and repeating for 3 treatment groups. And then carrying out culture experiments for 8 weeks, and after the experiments are finished, determining the residual eating behaviors of the fish, wherein the residual eating behaviors are judged according to whether the tail fin part of the takifugu rubripes has the bite marks. During cultivation, natural illumination and running water cultivation are adopted, the water temperature is 22-27 ℃, the salinity is 30-31, the pH is 7.5-8.5, and the dissolved oxygen is 7-8 mg/L.
(3) Sample collection and biochemical analysis
After the experiment is finished, all the juvenile fishes of the takifugu rubripes are subjected to starvation treatment for 24h, then each barrel of fishes is counted, whether biting traces exist or not is observed, and the starvation rate of the fishes is calculated.
(4) Statistical analysis
The experimental data are expressed as mean ± sem, and the one-way anova was performed with SPSS16.0, with P <0.05 as a significant difference.
(5) Results of the experiment
As can be seen from fig. 1, fig. 2 and fig. 3, the cannibalism rate of the takifugu rubripes shows a trend of first decreasing and then increasing along with the increase of the lysine, methionine or cysteine level in the feed, and dose effects of secondary curves exist, and the cannibalism rate has the lowest points at 4.05%, 1.12% and 2.32% of the lysine, methionine and cysteine levels in the feed respectively, which indicates that the suitable dose of the 3 amino acids added into the feed can effectively reduce the cannibalism rate of the takifugu rubripes; as can be seen from fig. 4 and 5, the cannibalism rate of the takifugu rubripes tends to be gentle after rising with the level of the feed DMPT or tryptophan, and the lowest point appears when the cannibalism rate is 0.030% for DMPT or 0.86% for tryptophan, which indicates that the cannibalism rate of the takifugu rubripes can be effectively reduced by a proper amount of DMPT or tryptophan.
Example 2 confirmatory screening of Complex additives under indoor Industrial flow-through aquaculture conditions
(1) Design of experiments
On the basis of a series of previous researches on reducing the residual eating behavior of the takifugu rubripes, relevant research achievements are integrated to carry out the researches. The specific experiment design is that commercial feed of the takifugu rubripes is used as the control group feed, the crude protein content of the commercial feed is 48%, and the crude fat content is 10%; the experimental group feed is prepared by adding different gradients of the compound additive for further verification screening on the basis of a control group feed. Based on the previous experimental results, 3 screening gradients were set, and the specific validated screening design recipe is shown in table 2.
TABLE 2 composite additive verification screening (% feed dry matter) to reduce cannibalism behavior of Fugu rubripes
Control group Experimental group L Experimental group M Experimental group H
Commercial feed 100 99.27 96.97 93.97
Lysine * 0 0.2 1.0 2
Methionine * 0 0.1 0.3 0.6
Cysteine* 0 0.3 1.2 2.4
DMPT* 0 0.03 0.03 0.03
Tryptophan * 0 0.1 0.5 1
Chemical composition
Crude protein 48.2 48.1 49.0 49.8
Crude fat 10.3 10.0 9.8 9.4
Lysine# 3.08 3.24 4.05 5.06
Methionine# 0.95 1.02 1.12 1.54
Cysteine# 1.24 1.51 2.32 3.61
DMPT# 0 0.03 0.03 0.03
Tryptophan# 0.37 0.43 0.86 1.33
Note: lysine, methionine, cysteine and tryptophan are all L-shaped, and the purity is more than 99%; DMPT with purity of over 98%. "+" indicates the added levels of lysine, methionine, cysteine, DMPT and tryptophan in the feed. "#" indicates the actual content of lysine, methionine, cysteine, DMPT and tryptophan in the feed.
(2) Experimental fish and breeding management
Before the experiment, the fugu rubripes which eats fresh rough fishes is domesticated by bait transferring for 4 weeks, so that the fugu rubripes is suitable for eating compound feed. Then, in order to make the eating behavior of the fugu rubripes in the cultivation process easy to observe, all the fishes for cultivation are not subjected to the tooth shearing treatment. After finishing bait turning, hungry for 24h, beginning formal breeding experiments, randomly putting 600 pieces of fugu rubripes with initial weight of about 30g into 12 breeding barrels (square, volume of 0.7m × 0.7m × 0.4m), putting 50 pieces of fugu rubripes into each barrel, and repeating 3 treatment groups. During the experiment, the mutual eating condition of the takifugu rubripes is counted for 1 time every 2 weeks, the eating behavior is judged according to whether the tail fin part of the takifugu rubripes has bite marks or not, the eating statistics comprises the sum of injured and dead fishes at the end of every 2 weeks, and the statistics is counted for 1 time every 2 weeks. The culture experiment period is 8 weeks, during which natural light irradiation and running water culture are adopted, the water temperature is 22-27 ℃, the salinity is 30-31, the pH is 7.5-8.5, and the dissolved oxygen is 7-8 mg/L.
(3) Sample collection and biochemical analysis
After the experiment is finished, all the juvenile fishes of the takifugu rubripes are subjected to starvation for 24h, and then each barrel of fishes is counted for calculating the survival rate of the fishes. Then, randomly taking 3 fishes from each barrel, carrying out excessive anesthesia by using eugenol, dissecting and taking the brains of the fishes, quickly freezing by using liquid nitrogen, and storing in a refrigerator at the temperature of-80 ℃ for measuring the content of 5-hydroxytryptamine.
The method for measuring 5-hydroxytryptamine content in brain comprises accurately weighing brain tissue by high performance liquid chromatography/electrochemical detection method, adding 0.5mL of pre-cooled trichloroacetic acid solution (2%) per 100mg of brain tissue, homogenizing on ice, and adding 0.2mL of NaH per 100mg of brain tissue2PO4Solution (concentration 0.33mol/L, pH 6, and Na content 0.5mol/L2EDTA) washing the homogenizer, and the washing solution is merged into the tissue homogenate, after vortex mixing, centrifugation is carried out for 20min at 4 ℃ at 10000g/min, and the supernatant is taken out and is subjected to mechanical determination.
(4) Statistical analysis
Statistical analysis of experimental data was the same as in example 1.
(5) Results of the experiment
As can be seen from FIG. 6, the content of 5-hydroxytryptamine in the brain of fugu rubripes in the control group is only 28.21ng/mg pro, which is lower than that in all experimental groups, and thus, the content of 5-hydroxytryptamine in the brain of fugu rubripes can be effectively increased, the pleasure mood of fish can be enhanced, and the behavior of attacking cannibalism can be reduced by using the compound additive provided by the invention. The conclusion is shown in the results in table 7, that the residual eating behavior of the takifugu rubripes is reduced by adding the compound additive compared with the control group, and the residual eating rate of the experimental group M and the experimental group H is remarkably reduced in 2-8 weeks, particularly the residual eating rate of the experimental group M is reduced by 31.3% compared with that of the control group in 8 weeks of cultivation, which indicates that the compound additive provided by the invention has the effect of remarkably reducing the residual eating behavior of the takifugu rubripes. As can be seen from fig. 3, the survival rate of the experimental group M in the 8 th week of cultivation is 91.3%, which is significantly higher than the survival rate of the control group of 68.7%, and thus the survival rate of the takifugu rubripes cultivation is improved by reducing the cannibalism behavior. Combining the results of table 3 and fig. 6 and 7, and combining the economic benefits, it was established that the actual contents of the composite additive of the present invention in the feed were "lysine 4.05%, methionine 1.12%, cysteine 2.32%, DMPT 0.03%, and tryptophan 0.86% dry matter of feed", respectively.
TABLE 3 mutual cannibalism of Fugu rubripes (%)
Control group Experimental group L Experimental group M Experimental group H
2 weeks 43.2±2.5a 38.0±3.2a 15.1±2.3b 18.4±2.1b
4 weeks 42.1±1.7a 36.9±2.4a 13.7±2.9b 19.2±1.7b
6 weeks 40.4±2.1a 32.8±2.1b 10.9±1.5d 17.3±1.4c
8 weeks 38.1±2.0a 30.7±1.9b 6.8±0.5d 16.1±1.3c
Example 3 Pilot-scale application Effect evaluation of Compound additive for decreasing residual eating behavior in winter Cross-season under indoor Industrial culture conditions
(1) Design of experiments
In the experiment, commercial feed is selected as a control group feed, the crude protein content of the commercial feed is 48%, and the crude fat content is 10%; the experimental group feed was prepared by adding the compound additives screened in example 2 on the basis of commercial feed, and the compound additive combinations and contents were "1.0% lysine + 0.3% methionine + 1.2% cysteine + 0.03% DMPT + 0.5% tryptophan", and the actual contents were "lysine 4.05%, methionine 1.12%, cysteine 2.32%, DMPT 0.03% and tryptophan 0.86% dry matter of feed", respectively.
(2) Experimental fish and breeding management
Before the start of the experiment, the fugu rubripes was acclimatized by bait transfer for 4 weeks as in example 2 so that it was adapted to ingest a compound feed, and all fish used for cultivation were not subjected to the treatment of clipping. After finishing bait transferring, hungry for 24h, beginning formal cultivation experiments, putting 2000 pieces of fugu rubripes with initial weight of about 200g into 2 cement ponds (square, volume of 2.5m × 2.5m × 1.5m), putting 1000 pieces of fish into each cement pond, and carrying out overwintering cultivation experiments in 2 months in indoor cement ponds, wherein the cultivation conditions during the overwintering period simulate the actual overwintering cultivation conditions of the fugu rubripes, namely, the water temperature of cultivation water is maintained at 16-18 ℃, the salinity is 18-20, the pH is 7.0-8.0, and the dissolved oxygen is 6-7 mg/L.
(3) Sample collection and biochemical analysis
And (3) counting the residual eating behaviors every 1 month during the culture period, judging the residual eating behaviors, randomly extracting 100 fishes, counting the marks of bitten at the tail fin part, specifically, randomly extracting 1 time in the morning, the noon and the evening, and taking the average value of 3 times as the final result. And (4) counting survival rate according to the number of dead fish recorded during the culture period and the mantissa of the fish put into the cement pond. The determination of 5-hydroxytryptamine content in brain is carried out by taking 10 Takifugu rubripes at random, anesthetizing with eugenol, and taking brain tissue, and determining 5-hydroxytryptamine by the same method as in example 1.
(4) Statistical analysis
The 5-hydroxytryptamine content and cannibalism rate in the brain of takifugu rubripes are expressed by mean value + -SE, and tested by SPSS16.0 for T test, and the difference is expressed by P < 0.05. The survival rate is not parallel, only the relative value is calculated, and statistical analysis is not carried out.
(5) Results of the experiment
As can be seen from the results in FIG. 8, similar to the results in example 2, the 5-hydroxytryptamine content in the fugu rubripes brain in the experimental group was 10.40ng/mg pro (P <0.05) and 13.12ng/mg pro (P <0.05) higher than that in the control group at month 1 and month 2 of cultivation, respectively; meanwhile, the residue food condition statistics of the takifugu rubripes also reflects the change trend consistent with the content of 5-hydroxytryptamine in fish brains, and the residue food rate in the experimental group is respectively reduced by 11.3% (P <0.05, figure 4) and 12.4% (P <0.05) in the 1 st month and the 2 nd month of the culture of the control group; finally, the survival rate was further counted and found to be 2.0% and 7.0% higher for the control composition than for the experimental group at 1 and 2 months after ingestion, respectively. The experimental results are combined, and the comprehensive nutrition method of the composite additive provided by the invention can effectively reduce the cannibalism behavior of the fugu rubripes under the classical overwintering cultivation condition and improve the cultivation survival rate.
Example 4 Pilot-scale application effect evaluation of composite additives under offshore cage culture conditions
(1) Design of experiments
In the experiment, commercial feed is selected as a control group feed, the crude protein content of the commercial feed is 48%, and the crude fat content is 10%; the experimental group feed was prepared by adding the compound additives selected in example 2 on the basis of commercial feed, and the compound additive combination and contents were "0.7% lysine + 0.2% methionine + 0.9% cysteine +0.03DMPT + 0.3% tryptophan", and the actual contents were "lysine 3.73%, methionine 1.05%, cysteine 2.12%, DMPT 0.03% and tryptophan 0.77% dry matter of feed", respectively.
(2) Experimental fish and breeding management
Before the start of the experiment, the fugu rubripes was acclimatized by bait transfer for 4 weeks as in example 2 so that it was adapted to ingest a compound feed, and all fish used for cultivation were not subjected to the treatment of clipping. After the bait is changed, hungry is carried out for 24h, formal culture experiments are started, namely, the takifugu rubripes with the initial weight of about 350g of 2500 fish is placed into 2 net cages (square, the volume is 3m multiplied by 2.5m), 1250 fish are placed into each net cage, the culture experiments of 5 months and 25 months are carried out in the offshore net cages from 5 days to 10 days, and the culture conditions of 5 months at the marine net cage culture stage are simulated, namely the water temperature of the culture water body is 20-28 ℃, the salinity is 31-32, the pH is 7.0-8.0, and the dissolved oxygen is 6-7 mg/L.
(3) Sample collection and biochemical analysis
The sample collection and biochemical analysis were the same as in example 4.
(4) Statistical analysis
The data were analyzed statistically as in example 4.
(5) Results of the experiment
The takifugu rubripes is cultured for 5 months, and the amount of 5-hydroxytryptamine in brain is 13.84ng/mg pro (P <0.05, FIG. 11) higher than that in the control group when the test group is fed with feed. Meanwhile, the rate of food retention was reduced by 7.9% (P <0.05, fig. 12) in the experimental group compared to the control group, while the rate of survival was increased by 4.6% (P <0.05, fig. 13). The results further show that the compound additive used in the method is also suitable for offshore cage culture conditions in summer, and can play a role in reducing the residual food of the takifugu rubripes and improving the culture survival rate.

Claims (5)

1. The compound additive for reducing the eating behavior of the takifugu rubripes is characterized by comprising lysine, methionine, cysteine, DMPT and tryptophan.
2. The takifugu rubripes feed added with the compound additive is characterized in that for the takifugu rubripes feed with the weight of 30-300g under the overwintering indoor culture condition, the culture water temperature is maintained at 16-18 ℃, the compound additive is added into the feed on the basis of commercial feed, and the mass ratio of each component of the compound additive in the feed to dry matter of the feed is respectively 4.05% of lysine, 1.12% of methionine, 2.32% of cysteine, 0.03% of DMPT and 0.86% of tryptophan.
3. The takifugu rubripes feed added with the compound additive is characterized in that under the condition of marine cage culture of takifugu rubripes with weight of 300-1000g in summer, the culture water temperature is 20-28 ℃, and the compound additive in the claim 1 is added on the basis of commercial feed, so that the mass ratio of each component of the compound additive in the feed to dry matter of the feed is 3.73% of lysine, 1.05% of methionine, 2.12% of cysteine, 0.03% of DMPT and 0.77% of tryptophan respectively.
4. The additive package according to claim 1, wherein said lysine, methionine, cysteine and tryptophan are all in L-form and have a purity of 99% or more; DMPT with purity of over 98%.
5. The takifugu rubripes feed according to any one of claims 2 or 3, wherein the lysine, methionine, cysteine and tryptophan are all in L-form and have a purity of 99% or more; DMPT with purity of over 98%.
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