A nutritional method and feed for improving the acute hypoxia tolerance of tetrosphicnus obscurus

Abstract

The application discloses a kind of nutrition method and feed for improving acute hypoxia tolerance of redfin pufferfish, belongs to the field of aquatic animal nutrition.The method uses the starch with the mass ratio of amylopectin and amylose being 2.41-2.49 as sugar source in the feed of redfin pufferfish, which can significantly improve the starch digestion rate of fish body, increase the adhesion of feed, reduce the loss of feed in water;At the same time, the expression of three hif alpha genes in liver and oxygen consumption are reduced, and the composition of intestinal flora is improved;Under hypoxia, the use of the feed of the application can promote the expression of hif-3 alpha gene in liver, better activate anaerobic glycolysis to provide energy for fish body, thereby improving the survival rate of redfin pufferfish under acute hypoxia.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of aquatic animal nutrition, and particularly relates to a nutritional method and feed for improving acute hypoxia tolerance of Takifugu rubripes. BACKGROUND

[0002] In natural waters and aquaculture, the dissolved oxygen content of the water environment is affected by many factors such as temperature, atmospheric pressure, day-night alternation, eutrophication, high-density culture, power supply state and live long-distance transportation, and is in a state of constant fluctuation. Sudden decrease in water body dissolved oxygen content will cause acute hypoxia stress in fish, affecting the growth, development and survival of fish, and causing significant economic losses. Therefore, finding ways to improve the hypoxia tolerance of aquatic animals is crucial for the healthy development of aquaculture. Hypoxia can cause energy deficiency and oxidative damage in fish. The common methods to improve the hypoxia tolerance of fish are to breed new varieties of hypoxia-tolerant fish or add antioxidants to the feed to improve the overall health status of fish. However, few researchers have improved the tolerance of fish to acute hypoxia by regulating nutritional metabolism.

[0003] A large number of studies have shown that fish will activate the hypoxia-inducible factor (HIF) and anaerobic glycolysis signaling pathway under acute hypoxia, decompose glucose to produce lactic acid and ATP, and at the same time reduce oxygen consumption and provide energy for the fish body. Adding an appropriate amount of carbohydrates (sugars) to the feed can save the use of fish meal and fish oil, reduce feed costs, and improve the water resistance and stability of feed particles. However, unlike mammals, most fish have weak ability to utilize carbohydrates (sugars) in feed. After glucose tolerance test or feeding high-sugar feed, fish continuously present a high blood sugar state, causing metabolic disorders and harming fish health. For complex sugars such as starch, they need to be decomposed by intestinal digestive enzymes before entering the blood, and by increasing the time of digestion and absorption in the intestine, fish have enough time to up-regulate the enzyme activity of related sugar metabolism, thereby promoting the utilization of sugars by fish. Starch is divided into amylose and amylopectin due to different polymerization ways. Amylose is connected by α-1,4 glycosidic bonds, while amylopectin is branched in the form of dendritic branches by α-1,6 glycosidic bonds on the basis of amylose, and has a more complex structure. Common sugar sources in fish feed include wheat starch, corn starch, cassava starch, potato starch, etc. The proportion of amylose and amylopectin in different starches is different, and the digestion and absorption rates also differ greatly. Takifugu rubripes is a marine carnivorous fish with degenerated gill cover and small gill pores. It will die in large quantities when the water dissolved oxygen content is lower than 1.5 mg / L. Therefore, there is an urgent need for a nutritional method to improve the acute hypoxia tolerance of Takifugu rubripes. SUMMARY

[0004] The technical problem solved by the present application is to provide a nutritional method and feed for improving the acute hypoxia tolerance of Takifugu rubripes.

[0005] The present application is implemented according to the following technical scheme:

[0006] The nutritional method for improving the acute hypoxia tolerance of Takifugu rubripes comprises adding 23% of starch by mass percentage in the feed of Takifugu rubripes, and the mass ratio of amylopectin to amylose in the starch is 2.41-2.49.

[0007] As one of the preferred embodiments, the starch is cassava starch.

[0008] As one of the preferred embodiments, the starch is mixed starch composed of amylopectin and amylose.

[0009] The feed for improving the acute hypoxia tolerance of Takifugu rubripes comprises fish meal and soybean protein concentrate as protein sources, fish oil and soybean lecithin as fat sources, and starch as sugar source, and the nutritional ingredients and the measured dry matter content are as follows: crude protein 44.25%-44.93%, crude fat 9.5%-9.81%, and the mass ratio of amylopectin to amylose in the starch is 2.41-2.49.

[0010] As one of the preferred embodiments, the starch is cassava starch.

[0011] As one of the preferred embodiments, the starch is mixed starch composed of amylopectin and amylose, and the mass ratio of amylopectin to amylose is 2.41-2.49.

[0012] As one of the preferred embodiments, the components and mass percentage of the feed are as follows: fish meal 42%, soybean protein concentrate 20%, starch 23%, fish oil 7%, soybean lecithin 1%, vitamin premix 1%, mineral premix 0.5%, choline chloride 0.5%, dibutyl hydroxytoluene 0.02%, dimethyl-beta-propionic acid thionine 0.1%, calcium dihydrogen phosphate 1%, vitamin C 0.5%, yttrium sesquioxide 0.1%, carboxymethyl cellulose 2%, cellulose 1.28%, and the starch is cassava starch or mixed starch composed of amylopectin and amylose according to the mass ratio of 2.41-2.49.

[0013] The present application has the following beneficial effects compared with the prior art:

[0014] (1) The starch with a mass ratio of 23% is added in the feed of Takifugu rubripes, so that the nutritional requirements of Takifugu rubripes for carbohydrates can be met, and the adhesion and water resistance of feed particles can be significantly improved.

[0015] (2) The application finds that, under the premise of not affecting growth performance, the use of cassava starch as a sugar source can significantly improve the sugar digestion rate and utilization rate of Takifugu rubripes in feed, thereby improving the acute hypoxia tolerance of Takifugu rubripes, and is significantly better than wheat starch and corn starch.

[0016] (3) The application artificially compounds mixed starches with two different proportions of amylopectin and amylose to simulate the proportions of amylopectin and amylose in corn starch and cassava starch, and finds that the acute hypoxia tolerance of Takifugu rubripes in the cassava starch simulation group is significantly higher than that in the corn starch simulation group. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 Effects of different sugar source feeds on acute hypoxia tolerance of Takifugu rubripes;

[0018] Figure 2 Effects of different sugar source feeds on weight gain rate of Takifugu rubripes;

[0019] Figure 3 Effects of different sugar source feeds on starch digestion rate of Takifugu rubripes;

[0020] Figure 4 Effects of different sugar source feeds on liver glycogen content of Takifugu rubripes;

[0021] Figure 5 Effects of different sugar source feeds on serum glucose content of Takifugu rubripes;

[0022] Figure 6 Effects of different sugar source feeds on serum pyruvic acid content of Takifugu rubripes;

[0023] Figure 7 Effects of different sugar source feeds on serum lactic acid content of Takifugu rubripes;

[0024] Figure 8 Effects of different sugar source feeds on liver lactic acid content of Takifugu rubripes;

[0025] Figure 9 Effects of different sugar source feeds on hif-1 alpha gene expression in livers of Takifugu rubripes;

[0026] Figure 10 Effects of different sugar source feeds on hif-2 alpha gene expression of Takifugu rubripes;

[0027] Figure 11 Effects of different sugar source feeds on hif-3 alpha gene expression of Takifugu rubripes;

[0028] Figure 12 Effect of amylopectin and amylose ratio on acute hypoxia tolerance of red puffer;

[0029] Figure 13 Effect of amylopectin and amylose ratio on weight gain rate of red puffer;

[0030] Figure 14 Effect of amylopectin and amylose ratio on starch digestibility of red puffer;

[0031] Figure 15 Effect of amylopectin and amylose ratio on serum lactic acid content. Specific embodiments

[0032] The technical solutions of the present application are further illustrated below through specific examples, but the protection scope of the present application is not limited in any form by the examples.

[0033] Example 1

[0034] This example provides the feed formula of different experiments, respectively adding different starches, and the specific composition and mass percentage are shown in Table 1.

[0035] Table 1 Feed formula of different sugar source experiments (% dry matter)

[0036]

[0037]

[0038] Note: 1 Vitamin premix (mg / g mixture): thiamine, 2.5 mg; riboflavin, 4.5 mg; vitamin B6, 2 mg; vitamin B12, 0.01 mg; biotin, 0.12 mg; vitamin K3, 1 mg; inositol, 80 mg; pantothenic acid, 6 mg; nicotinic acid, 20 mg; folic acid, 2 mg; vitamin A, 3.2 mg; vitamin D, 0.5 mg; vitamin E, 12 mg; wheat middlings, 866.17 mg.

[0039] 2 Mineral premix (mg / g mixture): ferrous sulfate, 112.7 mg; sodium chloride, 100 mg; magnesium sulfate, 50 mg; zinc sulfate, 45.2 mg; manganese sulfate, 9.3 mg; copper sulfate, 3.7 mg; cobalt chloride, 0.4 mg; calcium iodate, 0.3 mg; sodium selenite, 0.1 mg; zeolite powder, 678.3 mg.

[0040] (1) Experimental design and acute hypoxia survival rate comparison

[0041] Red pufferfish were divided into four groups and fed with diets containing wheat starch, corn starch, cassava starch or potato starch (3 tanks for each starch, 30 fish per tank). After 8 weeks of rearing, the fish in each tank were weighed to calculate the weight gain rate. To compare the effects of different starches on acute hypoxia survival rate, 10 fish were randomly selected from each tank and transferred to a custom-made hypoxia device. The dissolved oxygen in the water decreased from 6.5 mg / L to 1.0 mg / L as the fish breathed and the hypoxia time passed. The number of dead fish in each group was observed and recorded every 2 hours under acute hypoxia, and the acute hypoxia survival rate of different starch groups was calculated.

[0042] (2) Sample collection and index determination

[0043] In this experiment, sampling was divided into normoxic sampling and hypoxic sampling. After 12 hours of fasting, 12 fish were selected from each treatment group (4 fish per tank), of which 6 were placed in a normal dissolved oxygen tank (DO = 6.5 ± 0.5 mg / L, normoxic group), and the other 6 were placed in a hypoxic device (DO = 2.5 ± 0.5 mg / L, hypoxic group). After 6 hours, samples were collected simultaneously, and serum, liver and muscle were quickly frozen in liquid nitrogen and stored in a -80°C refrigerator for later use. The contents of glucose, pyruvate and lactate in serum, and the contents of glycogen and lactate in liver were determined using commercial kits (Nanjing Jiancheng Biological Engineering Research). The absorbance was read using an enzyme label instrument (Tecan infinite M200, Switzerland) and the content was calculated. RNA in the liver was extracted using the RNAiso Plus kit (TaKaRa, Dalian), and the relative expression of target genes was determined by real-time fluorescent quantitative PCR technology. Intestinal contents were collected, and the DNA of intestinal flora was extracted. The v3-v4 region of bacterial 16s rRNA was sequenced by the Illumina miseq platform (Shanghai Meiji Biomedicine Technology Co., Ltd.), and the composition of intestinal flora was analyzed. In addition, feces were collected during rearing, and the starch digestion rate was calculated by measuring the yttrium element and starch content.

[0044] (3) Data analysis

[0045] All data across multiple treatment groups were analyzed using SPSS Statistics 25.0 software (IBM, USA) via one-way ANOVA. Results are expressed as mean ± standard error (Mean ± SEM). Different letters (a, b, c) represent significant differences between treatment groups (p < 0.05). Independent-samples t-tests were used to analyze differences between two treatment groups. # (p < 0.05), ## (p < 0.01), and ### (p < 0.001) represent significant differences between the normoxic and hypoxic groups, respectively.

[0046] (4) Experimental Results

[0047] As shown in Table 1, the ratios of amylopectin to amylose in the wheat starch, corn starch, cassava starch, and potato starch diets were 1.36, 0.94, 2.41, and 1.05, respectively. This indicates that corn starch had the highest amylose content, while cassava starch had the highest amylopectin content. The effects of adding different starches to the feed on the hypoxia tolerance of red-finned pufferfish are as follows: Figure 1 As shown, dissolved oxygen levels in the water decreased continuously with prolonged hypoxia. When dissolved oxygen dropped to 1.6 mg / L, the survival rate of red-finned pufferfish in the cassava starch group was significantly higher than that in the corn starch group and the potato starch group (p < 0.05). When dissolved oxygen dropped to 1.4 and 1.0 mg / L, the survival rate of red-finned pufferfish in the cassava starch group was significantly higher than that in the corn starch group (p < 0.05). This indicates that feeding red-finned pufferfish with corn starch rich in amylose reduces their tolerance to acute hypoxia, while feeding them with cassava starch rich in amylopectin can improve their tolerance to acute hypoxia.

[0048] like Figure 2 As shown, the addition of different starches to the feed had no significant effect on the weight gain rate of redfin pufferfish (p > 0.05), but the digestibility of corn starch by redfin pufferfish was significantly lower than that of the other three starches. Figure 3 The results of glucose metabolism are as follows: Figures 4-8As shown in Fig. 2, the liver glycogen content of the cassava starch group was significantly higher than that of the potato starch group (p<0.05) under normal oxygen conditions, and had no significant difference with the other two groups (p>0.05). Under hypoxia, the liver glycogen content of the cassava starch group and the corn starch group decreased significantly (p<0.05), and the serum glucose content of the four starch groups increased significantly after hypoxia (p<0.05). Under normoxia, the serum glucose content of the corn starch group was significantly higher than that of the other three groups (p<0.05), and there was no significant difference in serum pyruvate content among the four groups (p>0.05), but the serum pyruvate content of the corn starch group and the cassava starch group increased significantly after hypoxia (p<0.05). Under normoxia, the serum lactic acid content of the corn starch group was significantly higher than that of the other three groups (p<0.05), and the serum lactic acid content of the four starch groups increased significantly after hypoxia (p<0.05). The liver lactic acid content of the cassava starch group was significantly higher than that of the wheat and corn starch groups under normoxia and hypoxia (p<0.05), and the liver lactic acid content of the cassava starch group and the wheat starch group increased significantly after acute hypoxia (p<0.05). The above results show that the red-spotted puffer fish has the lowest digestion rate of corn starch, and feeding cassava starch can better activate anaerobic glycolysis and promote the utilization of sugar by fish.

[0049] The results of HIF signaling pathway expression are shown in Fig. 3. Figures 9-11 As shown in Fig. 3, under normoxia, the gene expression of hif-1α and hif-3α of the red-spotted puffer fish fed with cassava starch was significantly lower than that of the wheat and corn starch groups (p<0.05), and the gene expression of hif-2α also showed a decreasing trend (p>0.05). Under hypoxia, the gene expression of hif-3α of the cassava starch group was significantly higher than that of the other three starch groups (p<0.05). Compared with the normoxia group, acute hypoxia significantly increased the gene expression of hif-2α and hif-3α of the red-spotted puffer fish in the wheat starch, cassava starch and potato starch groups, but did not affect the gene expression of hif-1α, hif-2α and hif-3α of the corn starch group (p>0.05). The above results show that under normoxia, feeding cassava starch can reduce the expression of three hifα genes in the liver and the consumption of oxygen; under hypoxia, feeding cassava starch can promote the expression of hif-3α gene and better activate anaerobic glycolysis to provide energy for fish.

[0050] The results of intestinal flora sequencing are shown in Table 2. At the phylum level, Spirochaetota and Proteobacteria are the dominant flora in the intestine of red pufferfish, and the abundance of Campilobacterota, Fusobacteriota and Firmicutes bacteria is also above 1%. Compared with the wheat starch group and the corn starch group, the abundance of Spirochaetota and Campilobacterota bacteria in the intestine of the cassava starch group and the potato starch group is increased, and the abundance of Proteobacteria and Fusobacteriota bacteria is decreased. The results at the genus level are shown in Table 3. Compared with the wheat starch group and the corn starch group, the abundance of Brevinema, norank-f-Arcobacteraceae and Shewanella in the intestine of the cassava starch group and the potato starch group is increased, and the abundance of Vibrio and Cetobacterium shows a downward trend. The above results show that different starches can also affect the tolerance of red pufferfish to hypoxia by changing the composition of intestinal flora.

[0051] Table 2 Effect of different sugar source feeds on the intestinal flora of red pufferfish at the phylum level

[0052]

[0053] Table 3 Effect of different sugar source feeds on the intestinal flora of red pufferfish at the genus level

[0054]

[0055]

[0056] In summary, the digestibility of red pufferfish to corn starch (rich in amylose) is the lowest, and feeding corn starch can reduce the tolerance of red pufferfish to acute hypoxia. Under normoxia, feeding cassava starch (the ratio of amylopectin to amylose is 2.41) can reduce the expression of three hifα genes in the liver and oxygen consumption, and improve the composition of intestinal flora; under hypoxia, feeding cassava starch can promote the expression of hif-3α gene in the liver, better activate anaerobic glycolysis to provide energy for the fish body, thereby improving the tolerance of red pufferfish to acute hypoxia.

[0057] Example 2

[0058] In this example, pure amylopectin and amylose (purchased from Arlindo Biochemical Technology Co., Ltd.) were used to artificially compound two different mixed starches with different proportions of amylopectin and amylose to simulate the proportions of amylopectin and amylose in corn starch (0.94) and cassava starch (2.41). The specific composition and mass percentage are shown in Table 4.

[0059] Table 4 Formulation of experimental feed with different sugar sources (% dry matter)

[0060]

[0061]

[0062] Note: 1 Vitamin premix (mg / g mixture): thiamine, 2.5 mg; riboflavin, 4.5 mg; vitamin B6, 2 mg; vitamin B12, 0.01 mg; biotin, 0.12 mg; vitamin K3, 1 mg; inositol, 80 mg; pantothenic acid, 6 mg; nicotinic acid, 20 mg; folic acid, 2 mg; vitamin A, 3.2 mg; vitamin D, 0.5 mg; vitamin E, 12 mg; wheat middlings, 866.17 mg.

[0063] 2 Mineral premix (mg / g mixture): ferrous sulfate, 112.7 mg; sodium chloride, 100 mg; magnesium sulfate, 50 mg; zinc sulfate, 45.2 mg; manganese sulfate, 9.3 mg; copper sulfate, 3.7 mg; cobalt chloride, 0.4 mg; calcium iodate, 0.3 mg; sodium selenite, 0.1 mg; zeolite powder, 678.3 mg.

[0064] (1) Experimental design and comparison of acute hypoxia survival rate: The red-spotted puffer fish were divided into two treatment groups and fed with simulated corn starch and simulated cassava starch (3 tanks for each starch treatment group, with 30 fish in each tank). After eight weeks of cultivation, the fish in each tank were weighed to calculate the weight gain rate. To compare the effects of different starches on acute hypoxia survival rate, 10 fish were randomly taken from each tank and transferred to a custom-made hypoxia device. As the fish breathed and the hypoxia time passed, the water dissolved oxygen decreased from 6.5 mg / L to 1.0 mg / L. The number of deaths in each group was observed and recorded every 2 hours under acute hypoxia, and the acute hypoxia survival rate of different mixed starch groups was calculated.

[0065] (2) Sample Collection and Index Determination: Sampling in this experiment was divided into normoxic and hypoxic sampling. After a 12-hour fast, 12 fish (4 fish per tank) were selected from each treatment group. Six fish remained in tanks with normal dissolved oxygen levels (DO = 6.5 ± 0.5 mg / L, normoxic group), while the other six were placed in a hypoxic environment (DO = 2.5 ± 0.5 mg / L, hypoxic group). Samples were collected simultaneously after 6 hours, and serum was stored at -80℃ for later use. Serum lactate content was determined using a commercial reagent kit (Nanjing Jiancheng Bioengineering Research Institute), and absorbance was read and content calculated using an ELISA reader (Tecan Infinite M200, Switzerland). In addition, feces were collected during the rearing process, and starch digestibility was calculated by measuring yttrium and starch content.

[0066] (3) Data analysis: All data were analyzed using SPSS Statistics 25.0 software (IBM, USA). The results are expressed as mean ± standard error (Mean ± SEM). Independent-samples t-test was used to analyze the differences between the two treatment groups. * (p<0.05), ** (p<0.01), and *** (p<0.001) represent significant differences between the simulated corn starch group and the simulated cassava starch group, respectively; # (p<0.05), ## (p<0.01), and ### (p<0.001) represent significant differences between the normoxic group and the hypoxic group, respectively.

[0067] (4) Experimental Results: As shown in Table 4, this embodiment used pure amylopectin and amylose to artificially blend two different amylopectin-amylose ratios of mixed starch. The measured amylopectin-amylose ratios in the simulated corn starch and simulated cassava starch were 0.92 and 2.49, respectively, which are very close to the amylopectin-amylose ratios of the real corn starch and cassava starch in Example 1.

[0068] The effects of adding different proportions of amylopectin and amylose to feed on the hypoxia tolerance of redfin pufferfish, such as Figure 12 As shown, with the extension of hypoxia time, the dissolved oxygen level in the water continued to decrease, and the fish began to die. When the dissolved oxygen dropped below 1.4 mg / L, the survival rate of redfin pufferfish in the simulated cassava starch group was significantly higher than that in the simulated corn starch group (p < 0.05), indicating that feeding simulated corn starch rich in amylose would reduce the tolerance of redfin pufferfish to acute hypoxia, while simulated cassava starch rich in amylopectin could improve the tolerance of redfin pufferfish to acute hypoxia.

[0069] like Figure 13The weight gain rate of the group fed with the simulated cassava starch containing more amylopectin showed a tendency to increase compared with the group fed with the simulated corn starch containing more amylose, but there was no significant difference (p>0.05). Figure 14 The results showed that the red-spotted puffer fish had a significantly higher digestion rate of amylopectin than amylose. The serum lactic acid results were as follows: Figure 15 As shown, under normal oxygen conditions, there was no difference in serum lactic acid content between the simulated corn starch group and the simulated cassava starch group (p>0.05); but under low oxygen conditions, the serum lactic acid content of the simulated cassava starch group was significantly higher than that of the simulated corn starch group (p<0.05); the serum lactic acid content of the simulated cassava starch group under low oxygen conditions was significantly higher than that of the normal oxygen group (p<0.05). The above results show that the red-spotted puffer fish has a higher digestion rate of amylopectin, and artificial high-amylopectin feed can improve the tolerance of red-spotted puffer fish to acute hypoxia by promoting the utilization of sugar by the fish.

Claims

1. A feed for improving the acute hypoxia tolerance of Takifugu rubripes, characterized by comprising, The feed comprises fish meal 42%, soybean protein concentrate 20%, starch 23%, fish oil 7%, soybean soft phospholipid 1%, vitamin premix 1%, mineral premix 0.5%, choline chloride 0.5%, dibutyl hydroxytoluene 0.02%, dimethyl-beta-propionic acid thionine 0.1%, calcium dihydrogen phosphate 1%, vitamin C 0.5%, yttrium sesquioxide 0.1%, carboxymethyl cellulose 2%, cellulose 1.28%, and the starch is cassava starch or mixed starch composed of amylopectin and amylose in a mass ratio of 2.41-2.

49.

2. A nutritional method for improving the acute hypoxia tolerance of Takifugu rubripes, characterized by, The method is to feed the redfin puffer fish with the feed for improving acute hypoxia tolerance of redfin puffer fish according to claim 1.

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