Mastacembelus armatus feed containing Chinese pulsatilla root extract and preparation method thereof

Abstract

The invention belongs to the technical field of feed processing, and particularly relates to a mastacembelus armatus feed containing a Chinese pulsatilla root extract and a preparation method thereof. The mastacembelus armatus feed containing the Chinese pulsatilla root extract is mainly prepared from the following raw materials: white fish meal, extruded soybeans, modified starch, beer yeast, fish oil, soybean oil, soybean lecithin, choline chloride, vitamin C phosphate, monocalcium phosphate, L-alanine, a vitamin and mineral premix, the Chinese pulsatilla root extract and the like. The Mastacembelus armatus feed prepared by the invention not only can significantly improve the growth performance and feed utilization efficiency of Mastacembelus armatus, but also can enhance the defense barrier function of intestinal mucosa of Mastacembelus armatus and relieve intestinal stress injury, and can effectively solve the problem of intestinal inflammation in current Mastacembelus armatus culture. Meanwhile, the feed for the mastacembelus armatus can also improve the adaptive capacity of the mastacembelus armatus to environmental changes, improve the overwintering survival rate of the mastacembelus armatus and promote the development of the artificial breeding industry of the mastacembelus armatus.

Description

Technical Field

[0001] The present invention belongs to the technical field of feed processing, and particularly relates to a Mastacembelus aculeatus feed containing Pulsatilla chinensis extract and a preparation method thereof. Background Art

[0002] Mastacembelus aculeatus belongs to the Perciformes Mastacembelidae and is a benthic fish. It is mainly distributed in the Yangtze River and Pearl River systems in the south, inhabiting the bottom of rivers with stones or the deep pool stone crevices with waterweeds on the shore. Mastacembelus aculeatus is a fish that mainly feeds on animal baits and also ingests some benthic animals and humus. Its meat is tender and delicious, without intermuscular spines, and has high nutritional value, being deeply loved by consumers. Currently, due to the influence of factors such as overfishing and environmental pollution, the germplasm resources of wild Mastacembelus aculeatus are increasingly depleted.

[0003] Currently, there is no special artificial compound feed developed for the feeding habits and physiological structure of Mastacembelus aculeatus under artificial breeding conditions. Its feed is mainly fed with fresh live animal feeds such as Tenebrio molitor, earthworms or maggots, or eel compound feed. However, it is found after breeding that Mastacembelus aculeatus is prone to indigestion and enteritis, which seriously affects the survival rate of fry cultivation. Therefore, researching and developing a suitable special feed for Mastacembelus aculeatus is of great significance for the artificial breeding of Mastacembelus aculeatus.

[0004] Patent document CN107668316A discloses a compound feed for Mastacembelus aculeatus adult fish. The feed mainly consists of cod fish meal, pregelatinized starch, fermented soybean meal, enzymolyzed fish meal powder, frozen fish oil, minced feed, minerals, calcium lactate, choline chloride, lysine, compound multivitamins and taurine; among them, the minced feed is made by crushing and mixing fresh fish meat, squid meat, shrimp meat and shrimp oil. This compound feed for Mastacembelus aculeatus adult fish can replace biological baits such as bloodworms to complete the feed conversion process to meet the nutritional requirements of Mastacembelus aculeatus adult fish, has good palatability, good feed conversion effect, high feed conversion rate, can effectively improve the liver and intestine health of fish, relieve the pollution of the aquaculture water body, and promote the growth and development of Mastacembelus aculeatus adult fish.

[0005] Patent document CN111165686A discloses a special feed for domesticating wild Mastacembelus aculeatus. The special feed for domesticating wild Mastacembelus aculeatus is prepared by proportioning fish meal, rapeseed meal, wheat bran, table salt, sodium bicarbonate, soybeans, betaine, choline chloride, phytase, fish oil, multivitamins, L-alanine and Tenebrio molitor, and imitating the shape of live baits such as mealworms to make it into soft pellet feed; the size and shape of the soft pellet feed can very well meet the palatability of Mastacembelus aculeatus, thereby promoting the faster and better completion of the feeding habit conversion of Mastacembelus aculeatus.

[0006] However, most of the current Mastacembelus armatus feeds focus on the research of nutrition, palatability and feed conversion rate, but the enteritis disease caused by artificial breeding of Mastacembelus armatus has not been properly solved. Therefore, aiming at the limitation of carbohydrate digestion due to the carnivorous characteristics of Mastacembelus armatus, further optimizing the breeding environment and feed formula, strengthening the maintenance of the intestinal health of Mastacembelus armatus, and improving the breeding technology system of Mastacembelus armatus are the key problems that need to be solved urgently in the current artificial breeding of Mastacembelus armatus. Summary of the Invention

[0007] In order to solve the defects existing in the prior art, the present invention provides a Mastacembelus armatus feed containing Pulsatilla chinensis extract and a preparation method thereof. The present invention creatively uses the Pulsatilla chinensis extract as an additive to the Mastacembelus armatus feed to solve the intestinal inflammation problem faced in the current breeding of Mastacembelus armatus. The Mastacembelus armatus feed containing Pulsatilla chinensis extract provided by the present invention can not only promote the growth of Mastacembelus armatus, improve the meat quality, but also strengthen the maintenance of the intestinal health of Mastacembelus armatus, prevent and treat enteritis diseases, and at the same time reduce the use of chemical drugs, which conforms to the concept of green breeding and promotes the sustainable development of the artificial breeding industry of Mastacembelus armatus.

[0008] The present invention provides a Mastacembelus armatus feed containing Pulsatilla chinensis extract, which comprises the following components and their weight parts:

[0009] 42 - 46 parts of white fish meal, 15 - 20 parts of extruded soybean, 15 - 20 parts of modified starch, 1 - 3 parts of brewer's yeast, 6 - 8 parts of fish oil, 2 - 4 parts of soybean oil, 1 - 2 parts of soybean lecithin, 0.4 - 0.6 part of choline chloride, 0.4 - 0.6 part of vitamin C phosphate, 1 - 2 parts of calcium dihydrogen phosphate, 0.4 - 0.6 part of L-alanine, 1 - 3 parts of vitamin and mineral premix, and 0.01 - 0.12 part of additive, wherein the additive is Pulsatilla chinensis extract.

[0010] Further, the Mastacembelus armatus feed containing Pulsatilla chinensis extract comprises the following components and their weight parts:

[0011] 45 parts of white fish meal, 18 parts of extruded soybean, 15 - 20 parts of modified starch, 2 parts of brewer's yeast, 7 parts of fish oil, 3 parts of soybean oil, 1 part of soybean lecithin, 0.5 part of choline chloride, 0.6 part of vitamin C phosphate, 1 part of calcium dihydrogen phosphate, 0.5 part of L-alanine, 2 parts of vitamin and mineral premix, and 0.03 part of additive, wherein the additive is Pulsatilla chinensis extract.

[0012] Further, the Pulsatilla chinensis extract is an extract of the dried root of Pulsatilla chinensis.

[0013] Further, the preparation method of the modified starch is as follows:

[0014] Step A: Mix choline chloride and mannose, and stir evenly. The mass ratio of choline chloride to mannose is 2:1 to obtain a mixed solvent;

[0015] Step B: Add corn starch into the mixed solvent obtained in Step S1, stir evenly to obtain a starch suspension. Adjust the pH of the starch suspension to 7.5 - 8.5, gelatinize it at 90 - 95°C for 1 - 2 h. After the reaction, add absolute ethanol, filter by suction to obtain the precipitate, wash it with absolute ethanol, and dry it to obtain pregelatinized starch;

[0016] Step C: Take sodium alginate and add it into distilled water, stir evenly to obtain a mixed solution. Add the pregelatinized starch obtained in Step B into the mixed solution, stir at 40 - 45°C for 20 - 30 min, dry it, pulverize it, and pass through a 100 - mesh sieve to obtain the product.

[0017] Further, the mass concentration of corn starch in the mixed solvent in Step B is 0.8 - 1.2 wt%.

[0018] Further, the mass ratio of pregelatinized starch to sodium alginate in Step C is 1:(0.05 - 0.1).

[0019] Further, the additive further includes chitosan oligosaccharide.

[0020] Further, the mass ratio of pulsatilla chinensis extract to chitosan oligosaccharide in the additive is 1:(0.4 - 0.8).

[0021] Further, the mass ratio of pulsatilla chinensis extract to chitosan oligosaccharide in the additive is 1:0.6.

[0022] In addition, the present invention also provides a preparation method of the macrognathus aculeatus feed containing pulsatilla chinensis extract, including the following steps:

[0023] Step S1: Take half of the modified starch and premix it with vitamins, minerals, additives, vitamin C phosphate, fish oil, and soybean oil evenly to obtain mixture I. Then add 35 - 40% of the total mass of the above mixture I of water, stir evenly to obtain mixture II;

[0024] Step S2: Mix the remaining modified starch with white fish meal, expanded soybean, brewer's yeast, soy lecithin, choline chloride, calcium dihydrogen phosphate, and L - alanine evenly. Then add the mixture II obtained in Step S1, mix well to obtain mixture III. Then add 30% of the total mass of the above mixture III of water, make it into pellet feed, and dry it in the air to obtain the product.

[0025] The inventor has been committed to the research on the artificial breeding of Mastacembelus armatus. The Pulsatilla chinensis plant is rich in components such as lignans, triterpenic acids, and saponins, and its biological activities such as regulating apoptosis, anti-tumor, antibacterial, antioxidant, and anti-inflammatory have attracted the attention of the inventor. In order to solve the enteritis problem in the artificial breeding of Mastacembelus armatus, the inventor put forward the idea of applying the Pulsatilla chinensis extract to the feed additive for Mastacembelus armatus. Through research, it is found that the Pulsatilla chinensis extract can enhance the immunity of Mastacembelus armatus, reduce the incidence of enteritis in Mastacembelus armatus, and promote the growth of Mastacembelus armatus by acting such as scavenging free radicals, inhibiting inflammatory factors, and regulating the NF-κB signaling pathway.

[0026] Furthermore, the inventor has experimentally proved that the feed added with the Pulsatilla chinensis extract can significantly improve the growth performance and feed utilization efficiency of Mastacembelus armatus. At the same time, it can also improve the integrity of the intestinal villi of Mastacembelus armatus, increase the thickness of the intestinal muscular layer, improve the digestion and absorption ability of the intestine, accelerate the formation of the tight junction protein barrier in the intestine, promote intestinal health, enhance the defense barrier function of the intestinal mucosa, reduce intestinal stress injury, and can effectively solve the intestinal inflammation problem faced in the current breeding of Mastacembelus armatus.

[0027] Furthermore, in order to better solve the water resistance of aquatic feed and prevent the loss of effective components such as the Pulsatilla chinensis extract. The inventor modified the starch. Specifically, the corn starch was pre-gelatinized first, and then a specific sodium alginate was added and mixed to obtain the modified starch. The modified starch has the advantages of good bonding performance and high stability. It can not only significantly reduce the dispersion and dissolution of pellet feed in water, improve the water resistance of the feed, but also effectively protect the stability of feed additives and reduce the loss of effective components and nutrients.

[0028] In addition, the inventor found in the process of consulting the literature that chitosan oligosaccharide has the effects of enhancing immunity, promoting growth, improving water quality, and regulating intestinal flora in aquatic feed. In order to further improve the treatment effect of the Pulsatilla chinensis extract on enteritis in Mastacembelus armatus, the inventor used chitosan oligosaccharide and the Pulsatilla chinensis extract in combination. It was found through experiments that compared with the single-component group of the Pulsatilla chinensis extract, the combined use of the two did not have obvious advantages in the prevention and treatment of enteritis in Mastacembelus armatus. However, during the experiment, the inventor unexpectedly found that when chitosan oligosaccharide and the Pulsatilla chinensis extract were used in combination in a specific ratio, the two could synergistically improve the regulation ability and adaptability of Mastacembelus armatus to external temperature fluctuations, and could effectively improve the overwintering survival rate of Mastacembelus armatus.

[0029] Compared with the prior art, the Mastacembelus armatus feed containing the Pulsatilla chinensis extract provided by the present invention has the following advantages:

[0030] (1) The Mastacembelus armatus feed containing Pulsatilla chinensis extract provided by the present invention can not only significantly improve the growth performance and feed utilization efficiency of Mastacembelus armatus, but also enhance the defense barrier function of the intestinal mucosa of Mastacembelus armatus, reduce intestinal stress injury, and effectively solve the intestinal inflammation problem faced in the current breeding of Mastacembelus armatus.

[0031] (2) The Mastacembelus armatus feed containing Pulsatilla chinensis extract provided by the present invention has good water resistance, can well maintain the content of active ingredients and vitamins in the feed, promote the absorption of nutritional components of Mastacembelus armatus, and is more conducive to the growth and reproduction of Mastacembelus armatus. Description of the Drawings

[0032] Figure 1 Diagram of the effect of adding different concentrations of Pulsatilla chinensis extract in the feed on the intestinal tissue of Mastacembelus armatus.

[0033] Figure 2 Diagram of the effect of adding different concentrations of Pulsatilla chinensis extract in the feed on the intestinal muscle layer thickness of Mastacembelus armatus.

[0034] Figure 3 Diagram of the effect of adding different concentrations of Pulsatilla chinensis extract in the feed on superoxide dismutase and malondialdehyde in the intestinal tissue of Mastacembelus armatus.

[0035] Figure 4 Diagram of the effect of adding different concentrations of Pulsatilla chinensis extract in the feed on the gene expression levels of related factors (IL-10, TNF-α, Occludin, Claudin-14, and Claudin-15) in Mastacembelus armatus.

[0036] Figure 5 Diagram of the effect of adding different concentrations of Pulsatilla chinensis extract in the feed on superoxide dismutase, malondialdehyde, and myeloperoxidase in the intestinal tissue of Mastacembelus armatus after Aeromonas veronii stress.

[0037] Figure 6 Diagram of the effect of adding different concentrations of Pulsatilla chinensis extract in the feed on the gene expression of TNF-α and IL-10 in the intestinal tissue of Mastacembelus armatus after Aeromonas veronii stress.

[0038] Figure 7 Diagram of the effect of adding different concentrations of Pulsatilla chinensis extract in the feed on the gene expression of Occludin, Claudin-14, and Claudin-15 in the intestinal tissue of Mastacembelus armatus after Aeromonas veronii stress.

[0039] Figure 8 Diagram of the effect of adding different concentrations of Pulsatilla chinensis extract in the feed on the histology of the intestinal tissue of Mastacembelus armatus after acetic acid-induced enteritis.

[0040] Figure 9Effect diagram of adding different concentrations of Pulsatilla chinensis extract to feed on the intestinal muscular layer thickness of Mastacembelus armatus after acetic acid-induced enteritis.

[0041] Figure 10 Effect diagram of adding different concentrations of Pulsatilla chinensis extract to feed on superoxide dismutase, malondialdehyde and myeloperoxidase in the intestinal tissue of Mastacembelus armatus induced by acetic acid.

[0042] Figure 11 Effect diagram of adding different concentrations of Pulsatilla chinensis extract to feed on the expression of TNF-α, IL-10, Occludin, Claudin-14 and Claudin-15 genes in the intestinal tissue of Mastacembelus armatus after acetic acid-induced enteritis. Detailed implementation mode

[0043] The present invention will be further described below through the description of specific implementation modes. However, this is not a limitation of the present invention. Those skilled in the art can make various modifications or improvements according to the basic idea of the present invention. As long as they do not depart from the basic idea of the present invention, they are all within the scope of the present invention. The materials and reagents involved in the present invention can be obtained through commercial purchase or conventional technical means in the art. For example, the vitamin and mineral premix is purchased from Guangdong Hainter Biotechnology Group Co., Ltd., and the Pulsatilla chinensis extract is purchased from Nanjing Daosifu Biotechnology Co., Ltd., with the CAS number of 84603-56-5.

[0044] I. Preparation of a Mastacembelus armatus feed containing Pulsatilla chinensis extract

[0045] 1. The formula of the Mastacembelus armatus feed containing Pulsatilla chinensis extract is shown in Table 1:

[0046] Table 1 Composition and nutritional components of Mastacembelus armatus feed containing Pulsatilla chinensis extract (g / kg)

[0047]

[0048] 2. Preparation method:

[0049] 2.1. The preparation method of the modified starch is as follows:

[0050] Step A: Mix choline chloride and mannose and stir evenly. The mass ratio of choline chloride to mannose is 2:1 to obtain a mixed solvent.

[0051] Step B: Add corn starch to the mixed solvent prepared in Step S1 and stir evenly. The mass concentration of corn starch in the mixed solvent is 1.0 wt%, to obtain a starch suspension. Adjust the pH of the starch suspension to 8.0, gelatinize it at 90°C for 2 h. After the reaction, add absolute ethanol, filter by suction to obtain the precipitate, wash it with absolute ethanol, and dry it to obtain pre-gelatinized starch.

[0052] Step C: Take sodium alginate and add it to distilled water, stir evenly. The mass ratio of the pregelatinized starch to sodium alginate is 1:0.08 to obtain a mixed solution. Add the pregelatinized starch prepared in Step B to the mixed solution, mix well, stir at a temperature of 40 °C for 25 min, dry, crush, and pass through a 100-mesh sieve to obtain the product.

[0053] 2.2 Preparation method of the Macrognathus aculeatus feed containing Pulsatilla chinensis extract:

[0054] Step S1: Take half of the modified starch and premix it with vitamins, minerals, additives, vitamin C phosphate, fish oil, and soybean oil evenly to obtain mixture I. Then add 35-40% of the total mass of the above mixture I of water and stir evenly to obtain mixture II.

[0055] Step S2: Mix the remaining modified starch with white fish meal, expanded soybean, brewer's yeast, soybean lecithin, choline chloride, calcium dihydrogen phosphate, and L-alanine evenly. Then add the mixture II prepared in Step S1, mix well to obtain mixture III. Then add 30% of the total mass of the above mixture III of water to make pellet feed, and dry it in the air to obtain the product.

[0056] Example 4: Collection and treatment of Macrognathus aculeatus samples

[0057] 1. Experimental grouping:

[0058] Add 0, 150, 300, and 600 mg / kg of Pulsatilla chinensis extract (group names are PC 0, PC 150, PC 300, and PC 600 respectively) to the basal diet. The basal diet and the feed preparation method refer to the preparation of the Macrognathus aculeatus feed containing Pulsatilla chinensis extract above.

[0059] 2. Experimental samples:

[0060] The large spiny eel fry for the experiment were provided by a large spiny eel farm in Guangzhou, Guangdong Province. Before the experiment, the control feed (0 mg / kg) was fed first. After being temporarily raised for two weeks, large spiny eel fry with healthy bodies and uniform sizes of 8 ± 2 cm were selected. They were randomly divided into 12 nylon nets with specifications of 1.5 m in length, 1.2 m in width, and 1.0 m in height. Among them, every 3 nets were fed the same kind of feed. The four sides and the four corners at the bottom were fixed with jars filled with sand, and a feeding tray was hung in each net. A sunshade was built above the cultivation net cage to avoid direct sunlight. They were fed twice a day (at 9:00 and 17:00) until apparent satiety for eight consecutive weeks. During the experiment, the water temperature was about 28 °C. After the feeding experiment ended, they were fasted for 24 h and then anesthetized with eugenol for sampling. Then, 3 fish were randomly selected from each cage to measure their body weight and body length, and the viscera and intestines were collected and weighed. Three intestinal samples were taken and placed in 4% formaldehyde, RNA preservation solution, and empty tubes respectively. The samples placed in 4% formaldehyde were stored in a refrigerator at 4 °C, and the other samples were placed in a refrigerator at -20 °C for analysis.

[0061] Experimental Example 1: Effects of Adding Pulsatilla chinensis Extract to Feed on the Growth Performance of Large Spiny Eel

[0062] 1. Experimental method:

[0063] In order to explore the effects of adding Pulsatilla chinensis extract to feed on the growth performance of large spiny eel, 0, 150, 300, and 600 mg / kg of Pulsatilla chinensis extract were added to the basal feed respectively, as shown in Table 1 specifically, and the experiment was carried out for eight weeks. After eight weeks of breeding, the large spiny eels were weighed and counted, and the weight gain rate (WGR), specific growth rate (SGR), feed conversion ratio (FCR), condition factor (CF), viscerosomatic index (VSI), hepatosomatic index (HSI), and survival rate (SR) of the large spiny eels were calculated.

[0064] The specific calculation formulas are as follows:

[0065] Weight gain rate (WG, %) = 100 × (Wt - W0) / W0;

[0066] Specific growth rate (SGR, % / d) = (LnWt - LnW0) / t × 100;

[0067] Feed conversion ratio (FCR) = WF / (Wm + Wd - Wn);

[0068] Condition factor (CF, g / cm3) = Wt / (L)3;

[0069] Viscerosomatic index (VSI, %) = Wv / Wt × 100%;

[0070] Hepatosomatic index (HSI, %) = Wh / Wt;

[0071] Survival rate (SUR, %) = [(N0 - Nt) / N0] × 100%;

[0072] Wherein, N0 and Nt respectively represent the number of Mastacembelus armatus at the start and at death, and Wt represents the average final body weight (g); W0 and Wt respectively represent the average values of the initial weight and the final weight of all Mastacembelus armatus (g); WF is the total feed intake (g); Wd is the weight of dead fish (g); Wh is the liver weight (g); Wm and Wn are respectively the total final weight and the initial total weight of Mastacembelus armatus; L is the body length of the sample fish (cm); Wv is the average weight of the visceral mass (g); t is the number of days of feeding (d).

[0073] 2. Experimental results:

[0074] The experimental results are shown in Table 2.

[0075] Table 2 Effects of adding Pulsatilla chinensis extract to the feed on the growth performance of Mastacembelus armatus

[0076]

[0077] Note: Different superscript letters in the same row indicate significant differences (p < 0.05), and the same letters indicate no significant differences (p > 0.05).

[0078] As can be seen from Table 2, the survival rate of the experimental fish can reach 100%. The weight gain rate and specific growth rate of PC 300 are the highest among other groups, and the viscerosomatic index and hepatosomatic index of this group are decreased compared with the control group. It can be seen that the concentration of Pulsatilla chinensis extract at 300 mg / kg has the best effect on improving the growth of Mastacembelus armatus. After adding Pulsatilla chinensis extract as a feed additive, the growth performance and feed utilization efficiency of Mastacembelus armatus have been significantly improved.

[0079] Specifically, for the group with 300 mg / kg of PC, its weight gain rate showed an obvious increase, but there was no significant effect (P>0.05), indicating that this dosage could more effectively promote the growth of fish. At the same time, the feed coefficient decreased significantly after adding the Pulsatilla chinensis extract, especially reaching the lowest value in the PC 300 group, suggesting that using the Pulsatilla chinensis extract as an additive could improve the feed conversion efficiency, resulting in better growth effects per unit of feed. Regarding the internal structure ratio of the fish body, the viscerosomatic index and hepatosomatic index decreased in each group with the addition of the Pulsatilla chinensis extract compared to the control group, although the degree of reduction was small, indicating that the Pulsatilla chinensis extract might have a positive effect on maintaining or optimizing the healthy size of internal organs, avoiding excessive growth, which is a beneficial factor for the long-term health and production performance of fish. In addition, the observed survival rate data highlighted the safety and adaptability of the Pulsatilla chinensis extract, with the survival rate of the experimental groups all reaching 100%, which also proved the safety of the Pulsatilla chinensis extract additive.

[0080] The above results show that: the Pulsatilla chinensis extract can significantly improve the growth performance and feed utilization efficiency of Mastacembelus armatus at a dosage of 300 mg / kg, while maintaining a high survival rate and good physiological health status.

[0081] Experimental Example 2: Effects of adding Pulsatilla chinensis extract to the feed on the intestinal histology of Mastacembelus armatus

[0082] 1. Experimental method:

[0083] To explore the effects of adding Pulsatilla chinensis extract to the feed on the intestinal histology of Mastacembelus armatus, 0, 150, 300, and 600 mg / kg of Pulsatilla chinensis extract were added to the basal feed respectively, as shown in Table 1 specifically, and the experiment was carried out for eight weeks. After eight weeks of cultivation, the intestines were subjected to HE staining. The specimens were placed in 4% formaldehyde solution and sent to Seville Company for paraffin embedding, sectioning, and HE staining. The sections were observed using a Nikon microscope, and the section images were analyzed using CapStudio and PS. Among them, according to (An Yuqi, "Suitable Proportion of Defatted Cottonseed Protein Complex Replacing Fish Meal in Monopterus albus Feed under the Condition of Unadded or Added Complex Enzyme Preparation", doi: 10.12418 / CJAN2023.721), the average value of the 8 longest villi measured in the section was used to measure the intestinal villus height; when measuring, the circular cross-section of the intestine in the section was divided into 8 parts along the "cross" shape at the center point to calculate the average value to measure the intestinal muscular layer thickness.

[0084] 2. Experimental results:

[0085] The experimental results are as Figure 1 and Figure 2 shown.

[0086] 2.1. The results of the effects of the Pulsatilla chinensis extract on the intestinal tissue of Mastacembelus armatus are as Figure 1As shown:

[0087] Figure 1 It is the diagram of the effect of adding different concentrations of Pulsatilla chinensis extract to the feed on the intestinal tissue of Mastacembelus armatus. Among them: A: PC 0, 40x; B: PC 150, 40x; C: PC 300, 40x; D: PC 600, 40x; a: PC 0, 100x; b: PC 150, 100x; c: PC 300, 100x; d: PC 600, 100x. From Figure 1 it can be seen that the structural integrity of the intestinal villi in the PC 300 group shows significant advantages, especially prominent compared with the control group and other experimental groups.

[0088] 2.2 The results of the effect of Pulsatilla chinensis extract on the intestinal muscular layer thickness of Mastacembelus armatus are as Figure 2 shown:

[0089] Figure 2 It is the diagram of the effect of adding different concentrations of Pulsatilla chinensis extract to the feed on the intestinal muscular layer thickness of Mastacembelus armatus. Among them: Different letters indicate significant differences, p < 0.05. After adding Pulsatilla chinensis extract to the feed for 8 weeks, from Figure 2 it can be seen that the intestinal muscular layer thickness of Mastacembelus armatus in the PC 150 and PC 300 groups increased significantly (P < 0.05). Notably, the thickening effect of PC 300 was more significant, exceeding that of PC 150. The above results show that PC 300 can improve the integrity of the intestinal villi of Mastacembelus armatus, increase the thickness of the intestinal muscular layer, improve the digestive and absorption capacity of the intestine, promote intestinal health, and play a certain role in the defense against diseases and stimuli.

[0090] From Figure 1 and Figure 2 it can be seen that the Pulsatilla chinensis extract in the feed additive for Mastacembelus armatus provided by the present invention can significantly improve the intestinal structure at 300 mg / kg and play a certain role in the defense against diseases and stimuli.

[0091] Experimental Example 3: Effect of adding Pulsatilla chinensis extract to the feed on the histochemistry of the intestine of Mastacembelus armatus

[0092] 1. Experimental method:

[0093] In order to explore the effect of adding Pulsatilla chinensis extract to the feed on the histochemistry of the intestine of Mastacembelus armatus, 0, 150, 300, and 600 mg / kg of Pulsatilla chinensis extract were added to the basal feed respectively, as shown in Table 1 specifically, and the experiment was carried out for eight weeks. After eight weeks of breeding, enzyme activity detection was carried out.

[0094] When measuring the protein concentration in tissues, the Beyotime Bradford Protein Assay Kit is usually used. A standard curve is obtained according to the usage method of the kit, and the protein concentration in the sample is determined by measuring the OD value of the processed sample. The standard curve shows that there is a good linear relationship between 5 μL of the sample or standard product in the concentration range of 0.1 - 1.5 mg / mL. Therefore, before starting the formal experiment, a preliminary experiment should be carried out to judge the sample dilution factor, so as to improve the accuracy of the experiment. Intestinal tissue homogenate was taken out from -80 °C and dissolved for later use. The kits provided by Nanjing Jiancheng Institute of Technology were used for testing, and the activities of SOD and the contents of MDA were tested respectively.

[0095] 1.1 Determination of superoxide dismutase (SOD):

[0096] The midgut tissue was taken out from the refrigerator at -80 °C and dissolved for later use. The kit provided by Nanjing Jiancheng Institute of Technology was used for testing. According to the standard that the SOD inhibition rate reaches 50% in 1 mL of the reaction solution per milligram of tissue protein, we determined the corresponding SOD amount as one SOD activity unit (U). The calculation formula:

[0097]

[0098] 1.2 Determination of malondialdehyde (MDA):

[0099] The midgut tissue was taken out from the -80 °C refrigerator for dissolution, and the kit provided by Nanjing Jiancheng Institute of Technology was used for determination. All reagents were added according to a specific reaction system. The calculation formula:

[0100]

[0101] 2. Experimental results:

[0102] The experimental results are as Figure 3 shown.

[0103] Figure 3 It is a diagram showing the effects of adding different concentrations of Pulsatilla chinensis extract in the feed on the superoxide dismutase (SOD) and malondialdehyde (MDA) in the intestinal tissue of Mastacembelus armatus, where different letters indicate significant differences, p < 0.05.

[0104] From Figure 3It can be seen that, compared with the control group, the superoxide dismutase (SOD) activity in the large spiny loach of PC 150 and PC 300 showed a significant downward trend (P<0.05), while the PC 600 group showed an extremely significant increase in SOD activity (P<0.05), indicating that the extracts of Pulsatilla chinensis at different concentrations have an obvious regulatory effect on the activity of SOD enzyme. The detection results of malondialdehyde (MDA) content showed that there was no statistically significant difference between PC 150 and the control group (P>0.05), suggesting that the extract of Pulsatilla chinensis has limited effect on lipid peroxidation at this concentration. However, the MDA content of PC 300 and PC 600 showed extremely significant reduction (P<0.05), indicating that the extract of Pulsatilla chinensis at higher doses can effectively inhibit lipid peroxidation reaction and reduce the damage of oxidative stress to organisms.

[0105] The above results show that at low concentration (PC 150), it may not significantly affect SOD activity or MDA level due to insufficient dose, while at medium and high concentrations (PC 300 and PC 600), it can effectively enhance SOD activity and significantly reduce MDA content, thus showing strong antioxidant and anti-lipid peroxidation abilities.

[0106] Experimental Example 4: Effects of adding Pulsatilla chinensis extract to feed on the intestinal immune function and intestinal barrier of large spiny loach

[0107] 1. Experimental method:

[0108] In order to explore the effects of adding Pulsatilla chinensis extract to feed on the intestinal immune function and intestinal barrier of large spiny loach, 0, 150, 300, and 600 mg / kg of Pulsatilla chinensis extract were added to the basal feed respectively, as shown in Table 1 specifically, and the experiment was carried out for eight weeks. After eight weeks of breeding, the intestinal tissues were taken to detect the mRNA expression of genes related to immunity and intestinal barrier.

[0109] Among them: The method for total RNA extraction and gene expression analysis is as follows:

[0110] For each group, the intestine was taken, 1 mL of TransZol Up (Transgen, Beijing) was added, and the tissue was broken at low temperature with a tissue grinder until there were no obvious particles. Total RNA was extracted from the intestine according to the TransZol Up instruction manual; the RNA concentration and purity were detected using a ultra-micro spectrophotometer (KAIAO K5600C, Beijing). cDNA synthesis was carried out using TransGen All-in-One First-Strand cDNA Synthesis SuperMix for qPCR (One-Step gDNA Removal) reagent. According to the steps in the instruction manual, reverse transcribe to synthesize the first strand of cDNA as the template for gene expression qPCR detection and store it at -80 °C for later use. Design specific primers using Primer Premier 5 software and synthesize them at Guangzhou Tianyi Huiyuan Gene Technology Co., Ltd. Use a real-time fluorescence quantitative gene amplifier (qPCR) (analytik jena qTOWER 3 , Germany) to perform qPCR detection on the related genes in the intestine (see Table 3). The 20 μL reaction system consists of 10 μL of SuperMix, 0.5 μL of each upstream and downstream primer, 4 μL of ddH2O, and 5 μL of cDNA. The procedure is as follows: pre-denaturation at 94 °C for 30 s; 94 °C for 5 s; 60 °C for 30 s; a total of 40 cycles; finally, the dissociation stage. Using the β-actin gene as an internal reference, quantitatively determine the mRNA expression levels of IL-10, TNF-α, Occludin, Claudin-14, and Claudin-15 in intestinal tissues by real-time quantitative PCR. Each sample has 3 replicates, and the relative expression levels of the genes are calculated using the 2-ΔΔCt method.

[0111] Table 3 Related genes in the intestine

[0112]

[0113] 2. Experimental results:

[0114] The experimental results are as Figure 4 shown.

[0115] Figure 4 Figure showing the effects of adding different concentrations of Pulsatilla chinensis extracts in the feed on the gene expression levels of related factors (IL-10, TNF-α, Occludin, Claudin-14, and Claudin-15) in Mastacembelus armatus. Among them, different letters indicate significant differences, p < 0.05.

[0116] From Figure 4It can be seen that the TNF-α gene expression levels in the intestinal tracts of Mastacembelus armatus in each group supplemented with Pulsatilla chinensis extract (PC 150, PC 300, PC 600) were significantly lower than those in the non-supplemented group (PC0) (P<0.05), and those in PC 300 and PC 600 were lower than those in PC 150. However, when comparing each group supplemented with Pulsatilla chinensis extract (PC 150, PC 300, PC 600) with the non-supplemented group (PC 0), although the IL-10 gene expression levels were also significantly decreased, those in PC 300 were close to those in the control group. As an anti-inflammatory factor, IL-10 effectively inhibits the synthesis of inflammatory factors.

[0117] The above results indicate that the inflammatory factor-related pathways in the intestinal tissues of Mastacembelus armatus in PC 300 were activated, resulting in a decrease in the expression level of the TNF-α gene in the midgut tissues, while the expression level of the IL-10 gene was close to that in the control group, thereby reducing inflammatory damage.

[0118] However, the expression levels of the tight junction protein genes Occludin and Claudin-14 in the intestine of PC300 were significantly increased compared with those in the control group (P<0.05), while the expression level of Claudin-15 was significantly decreased. The above results show that PC 300 accelerates the formation of the tight junction protein barrier in the intestine, enhances the defense barrier function of the intestinal mucosa, and reduces intestinal stress damage.

[0119] Experimental Example 5: Effects of Supplementing Pulsatilla chinensis Extract in Feed on the Intestine of Mastacembelus armatus after Aeromonas veronii Stress

[0120] 1. Model construction:

[0121] To explore the effects of supplementing Pulsatilla chinensis extract in feed on the intestine of Mastacembelus armatus after Aeromonas veronii stress, 0, 150, 300, and 600 mg / kg of Pulsatilla chinensis extract were added to the basal feed respectively, as shown in Table 1 specifically, and the experiment was carried out for eight weeks. After eight weeks of breeding, 10 fish were randomly selected from each group for Aeromonas veronii challenge test (the groups were named PC0, PC 150, PC 300, PC 600), and all fish were intraperitoneally injected with 50 μL of bacterial suspension (1x10 8 CFU / mL). As a negative control, 10 fish in the PC 0 group were only injected with normal saline (the group was named control, called the control group). Observe for 6, 24, and 48 h, carefully monitor the fish, and sample at the specified time points. Three fish were taken from each group, their intestines were taken after being anesthetized with eugenol, and one portion was placed in RNA protectant solution and one portion was put into an empty tube, both were placed at -80 °C for analysis.

[0122] 2. Experimental method:

[0123] 2.1. Determination of the effect of adding Pulsatilla chinensis extract to the feed on the intestinal histochemistry of Mastacembelus armatus after Aeromonas veronii stress:

[0124] The determination methods of the activity of SOD and the content of MDA were detected according to the experimental method in Experimental Example 3; the myeloperoxidase (MPO) was detected using a myeloperoxidase (MPO) activity detection kit (Beijing Boxbio Science & Technology Co., Ltd.).

[0125] 2.2. Determination of the effect of adding Pulsatilla chinensis extract to the feed on the intestinal immune function and intestinal barrier of Mastacembelus armatus after Aeromonas veronii stress:

[0126] The determination method was detected according to the experimental method in Experimental Example 4.

[0127] 3. Experimental results:

[0128] The experimental results are as Figure 5 and Figure 7 shown.

[0129] 3.1. The results of the effect of adding Pulsatilla chinensis extract to the feed on the intestinal histochemistry of Mastacembelus armatus after Aeromonas veronii stress are as Figure 5 shown:

[0130] Figure 5 It is the influence diagram of superoxide dismutase (SOD), malondialdehyde (MDA) and myeloperoxidase (MPO) in the intestinal tissue of Mastacembelus armatus after adding different concentrations of Pulsatilla chinensis extract to the feed under Aeromonas veronii stress. Among them: Different letters indicate significant differences, p < 0.05.

[0131] From Figure 5 it can be seen that in the initial stage after Aeromonas veronii stress, the SOD activity showed an upward trend, indicating that the body initiated an emergency antioxidant response to cope with the oxidative stress caused by bacterial infection. However, with the prolongation of the stress time, the SOD activity gradually decreased, which may reflect the adjustment of the body's antioxidant system regulation mechanism. It is worth noting that the PC 300 group still maintained a relatively high SOD activity 48 hours after stress. Compared with the control group, this significant antioxidant capacity highlights the positive role of PC300 in maintaining a long-term antioxidant defense state.

[0132] As a marker of lipid peroxidation, the MDA content increased significantly in the initial stage after stress, reflecting the degree of damage to the cell membrane caused by Aeromonas veronii infection. However, the MDA content in the PC 300 group 48 hours after stress was significantly lower than that in the control group and the model group. This result strongly proves that PC 300 can effectively inhibit the lipid peroxidation process, protect the cell membrane structure from oxidative damage, and thus reduce cell damage under stress conditions.

[0133] As a key indicator of the inflammatory response, the decreasing trend of myeloperoxidase (MPO) in the early stage of stress may reflect the early anti-inflammatory effect of PC 300, and the subsequent increase may be related to the body's immune response in the later stage. The MPO content in the PC 300 group was significantly higher than that in the control group 48 hours after stress. However, considering the changes in SOD activity and MDA content, this upregulation may reflect the complex mechanism of PC 300 in regulating the inflammatory response, that is, moderately stimulating immune function on the basis of protective antioxidant response to eliminate pathogens and promote tissue repair.

[0134] The above results indicate the regulatory effect of PC 300 on antioxidant and inflammatory responses under Aeromonas veronii stress. PC 300 can not only enhance the SOD activity in the early stage after stress, strengthen the body's antioxidant defense, but also maintain significant antioxidant capacity 48 hours after stress, effectively inhibit the MDA content, and reduce the degree of lipid peroxidation. In addition, the regulation of the MPO level by PC300 demonstrates its potential in balancing anti-inflammatory and immune responses, providing important clues for further understanding the application of additives in fish health management and disease prevention.

[0135] 3.2 Effects of adding Pulsatilla chinensis extract to the feed on the intestinal immune function of Mastacembelus armatus after Aeromonas veronii stress Figure 6 are shown as follows:

[0136] Figure 6 Figure showing the effects of adding different concentrations of Pulsatilla chinensis extract to the feed on the gene expression of TNF-α and IL-10 in the intestinal tissue of Mastacembelus armatus after Aeromonas veronii stress, where different letters indicate significant differences, p < 0.05.

[0137] From Figure 6 it can be seen that in the early stage after stress, the gene expression of TNF-α increased rapidly, which is consistent with the inflammatory cascade reaction triggered by bacterial infection. Although the TNF-α expression level in the PC 300 group was not the highest, the significant change in its expression compared with the control group reveals the complex role of PC 300 in regulating the level of pro-inflammatory cytokines, probably by inhibiting excessive inflammatory responses and promoting immune balance.

[0138] As an important anti-inflammatory mediator, at the time points of 6 hours and 24 hours after stress, the IL-10 expression level in the PC 300 group was significantly higher than that in the control group, indicating that PC 300 can effectively stimulate the anti-inflammatory response and reduce the inflammatory damage caused by Aeromonas veronii infection. At 48 hours after stress, the difference in IL-10 expression between the two groups decreased, which may reflect the role of PC 300 in maintaining long-term immune homeostasis, that is, rapidly inhibiting inflammation by upregulating IL-10 expression in the early stage of stress and then gradually restoring normal immune function.

[0139] 3.3 Effects of adding Pulsatilla chinensis extract to feed on the intestinal barrier of Mastacembelus armatus after Aeromonas veronii stress Figure 7 are shown as follows:

[0140] Figure 7 Figure showing the effects of adding different concentrations of Pulsatilla chinensis extract to feed on the expression of tight junction protein family genes Occludin, Claudin-14, and Claudin-15 in the intestinal tissues of Mastacembelus armatus after Aeromonas veronii stress. Among them, different letters indicate significant differences, p < 0.05.

[0141] It can be seen from Figure 7 that the expression of the tight junction protein family, including the genes Occludin, Claudin-14, and Claudin-15, is a key molecule in the formation of the intestinal epithelial barrier. After Aeromonas veronii stress, the expression of these genes shows a trend of first increasing and then decreasing, reflecting the immediate repair mechanism and subsequent recovery process of the intestine in response to infection. The significant effect of PC 300 on the expression of tight junction protein genes indicates that it plays an important role in enhancing intestinal barrier integrity, reducing pathogen invasion, and maintaining intestinal homeostasis.

[0142] Experimental Example 6: Effects of adding Pulsatilla chinensis extract to feed on acetic acid-induced enteritis in Mastacembelus armatus

[0143] 1. Model construction:

[0144] To explore the effects of adding Pulsatilla chinensis extract to feed on acetic acid-induced enteritis in Mastacembelus armatus, 0, 150, 300, and 600 mg / kg of Pulsatilla chinensis extract were added to the basal feed, as shown in Table 1 specifically, and the experiment was carried out for eight weeks. After eight weeks of breeding, 10 fish were randomly selected from each group for 3% acetic acid injection to construct a sampling model (group names are model, PC 150, PC300, PC 600, and the group with 0 mg / kg addition became the model group after acetic acid injection to construct the enteritis model). As a negative control, 10 fish in the PC 0 group were only injected with normal saline (group name is control, called the control group). After 24 h of model establishment, the fish were sampled. Three fish in each group were anesthetized with eugenol and their intestines were taken, and placed in 4% formaldehyde, RNA protective solution, and empty tubes respectively. The samples placed in 4% formaldehyde were stored at 4°C, and other samples were placed at -80°C for further analysis.

[0145] 2. Experimental methods:

[0146] 2.1 Determination of the effects of adding Pulsatilla chinensis extract to feed on intestinal histology after acetic acid-induced enteritis in Mastacembelus armatus:

[0147] The measurement method was carried out for detection with reference to the experimental method in Experimental Example 2.

[0148] 2.2. Determination of the effect of adding Pulsatilla chinensis extract in the feed on intestinal histochemistry after acetic acid-induced enteritis in Mastacembelus armatus:

[0149] The measurement methods for the activity of SOD and the content of MDA were carried out for detection with reference to the experimental method in Experimental Example 3; the myeloperoxidase (MPO) was detected using a myeloperoxidase (MPO) activity detection kit (Beijing Boxshengong Technology Co., Ltd.).

[0150] 2.3. Determination of the effect of adding Pulsatilla chinensis extract in the feed on intestinal immune function and intestinal barrier after acetic acid-induced enteritis in Mastacembelus armatus:

[0151] The measurement method was carried out for detection with reference to the experimental method in Experimental Example 4.

[0152] 3. Experimental results:

[0153] 3.1. The result diagrams of the effect of adding Pulsatilla chinensis extract in the feed on intestinal histology after acetic acid-induced enteritis in Mastacembelus armatus are as Figure 8 and Figure 9 shown:

[0154] Figure 8 are the diagrams of the effect of adding different concentrations of Pulsatilla chinensis extract in the feed on intestinal histology after acetic acid-induced enteritis in Mastacembelus armatus, where: A: control group, 40x; B: model group, 40x; C: PC 150, 40 times; D: PC 300, 40 times; E: PC 600, 40x; A: control group, 100x; B: model group, 100x; c: PC 150, 100x; d: PC 300, 100x; e: PC 600, 100x;. It can be seen from Figure 1 that compared with the control group and the model group, with the increase in the dose of Pulsatilla chinensis extract in the feed, especially PC 300, in Mastacembelus armatus induced with enteritis by acetic acid solution, the number of goblet cells increased significantly, and the villus length and villus integrity were significantly improved, and these indicators reached the optimal state at this dose.

[0155] Figure 9 is the diagram of the effect of adding different concentrations of Pulsatilla chinensis extract in the feed on the intestinal muscular layer thickness after acetic acid-induced enteritis in Mastacembelus armatus, where: different letters indicate significant differences, p<0.05. It can be seen from Figure 9 that in the PC 300 group after inducing enteritis by injecting acetic acid solution, the intestinal muscular layer thickness of Mastacembelus armatus increased significantly compared with the control group (P<0.05), indicating that this dose is beneficial to maintaining the normal intestinal structure.

[0156] It can be seen from Figure 8 and Figure 9It can be seen that the anemone extract additive in the Mastacembelus armatus feed at 300 mg / kg can significantly resist diseases caused by external factors, maintain the integrity of the intestinal structure, and play an obvious protective role in intestinal health.

[0157] 3.2. The results of the effects of adding anemone extract to the feed on the intestinal histochemistry of Mastacembelus armatus after acetic acid-induced enteritis are as Figure 10 shown:

[0158] Figure 10 Figure showing the effects of adding different concentrations of anemone extract to the feed on superoxide dismutase (SOD), malondialdehyde (MDA), and myeloperoxidase (MPO) in the intestinal tissue of Mastacembelus armatus induced by acetic acid. Among them: different letters indicate significant differences, p < 0.05. From Figure 10 it can be seen that when anemone extract is added, compared with the control, the SOD activity is increased, reflecting stronger antioxidant capacity. For the MDA content, compared with the control group, PC 150 and PC 300 are significantly decreased (P < 0.05). For the MPO content, compared with the control group, at the dose of 300 mg / kg, the MPO content is significantly increased (P < 0.05).

[0159] The above results indicate that within a certain dose range, the anemone extract can significantly change the antioxidant and inflammation-related indexes in the intestine of Mastacembelus armatus, and its effect varies with different doses.

[0160] 3.2. The results of the effects of adding anemone extract to the feed on the intestinal immune function and intestinal barrier of Mastacembelus armatus after acetic acid-induced enteritis are as Figure 11 shown:

[0161] Figure 11 Figure showing the effects of adding different concentrations of anemone extract to the feed on the expression of TNF-α, IL-10, tight junction protein family Occludin, Claudin-14, and Claudin-15 genes in the intestinal tissue of Mastacembelus armatus induced by acetic acid. Among them: different letters indicate significant differences, p < 0.05.

[0162] From Figure 11It can be seen that after acetic acid injection induced enteritis, the anti-inflammatory factors TNF-α and IL-10 were significantly upregulated in PC 300. At the same time, the expression of Occludin, Claudin-14, and Claudin-15 genes in the intestinal tissue of PC 300 was significantly upregulated, which accelerated the maturation and stability of the tight junction protein complex between intestinal epithelial cells, thereby significantly enhancing the intestinal mucosal physical barrier and improving the integrity and functionality of the intestinal mucosa. This enhancement can effectively reduce intestinal tissue damage caused by various stress factors, maintain the integrity of the intestinal barrier, reduce inflammatory reactions, thereby protecting the body from harmful substances, and maintaining the balance of intestinal microecology and overall health.

[0163] Experimental Example 7: Effects of Adding Pulsatilla chinensis Extract to Feed on Intestinal Health of Trimeresurus striata and Transcriptomics Analysis

[0164] 1. Experimental methods:

[0165] In order to explore the effect of adding Pulsatilla extract to feed on the intestinal health of giant spiny loach and transcriptomic analysis, 0 and 300 mg / kg of Pulsatilla extract were added to the basic feed, as shown in Table 1 (named PC 0 and PC300, respectively), and the experiment was cultured for eight weeks. Fasted for 24 hours before sampling, the viscera were separated on a 4℃ ice tray, and about 0.1g of the intestine was taken into a 1.5mL RNAse-free centrifuge tube and stored in a -80℃ refrigerator in time for transcriptomic analysis.

[0166] The intestinal tissue samples of the PC 0 and PC 300 treatment groups were stored in dry ice and sent to a sequencing company (Yuanxin Bio) for second-generation transcriptome sequencing. In normal species, more than 90% of RNA is rRNA. After extracting the total RNA of the sample, rRNA is removed by a conventional kit and mRNA is enriched. The enriched mRNA is reverse transcribed to form double-stranded cDNA. After repairing the double ends of the cDNA, a linker is added and PCR amplification is performed to construct an on-machine library. Specifically, the reference genome analysis, differential gene expression analysis and KEGG-based GSEA analysis were performed on the giant loach.

[0167] 2. Experimental results:

[0168] 2.1. Transcriptome sequencing results:

[0169] Using Illumina transcriptome sequencing technology, transcriptome sequencing was performed on the intestinal samples of Mastacembelus armatus fed with feed supplemented with Pulsatilla chinensis extract. There were a total of 3 control groups (PC 0) and 3 experimental groups (PC 300). After quality assessment and data filtering, 81.089 Gb of Clean Data was obtained. The average data volume of each sample's Clean Data was 10.136 Gb, the percentage of Q30 bases was above 95.9449%, and the GC content was between 46.9045% and 47.4654%. The quality-controlled sequencing data of each sample was aligned with the designated reference genome respectively, and the alignment rates ranged from 75.772% to 76.988%, among which the unique alignment rate was between 73.231% and 74.139%. A total of 24,809 genes were involved in the expression analysis, including 24,809 known genes (22,014 expressed) and 0 novel genes; there were 46,125 transcripts, including 46,125 known transcripts (36,010 expressed) and 0 novel transcripts. These results indicate that the sequencing data obtained in this study is highly reliable and can be used for further analysis.

[0170] 2.2. Screening of significantly differentially expressed genes

[0171] The results showed that: when Pulsatilla chinensis extract was added to the feed, PC 300 VS PC 0, there were 190 up-regulated genes and 173 down-regulated genes.

[0172] 2.3. GO and KEGG enrichment analysis of differentially expressed genes

[0173] The results showed that:

[0174] (1) Compared with the control group PC 0, in biological processes, biological regulation, cellular process, developmental process, immune system process, and metabolic process were significantly enriched; in cellular components, cell, cell part, membrane, and organelle were significantly enriched; in molecular functions, binding, catalytic activity, transporter activity, and transcription regulator activity were significantly enriched.

[0175] (2) The DEGs of the intestinal transcriptome with the addition of Pulsatilla chinensis extract in the feed were mainly enriched in pathways such as the intestinal immune network for IgA production, antigen processing and presentation, FoxO signaling pathway, and IL-17 signaling pathway, showing significant upregulation. Pathways such as retinol metabolism, glycine, serine, and threonine metabolism, and bile secretion showed significant downregulation.

[0176] 2.4, GSEA analysis based on KEGG

[0177] The results showed that among the top twenty significant pathways, there was a high overlap with the KEGG differential gene enrichment pathways. Combining the two parts of the pathways, it was found that the intestinal immune network for IgA production, tight junction, antigen processing and presentation, and FoxO signaling pathway were significantly upregulated, indicating that the addition of Pulsatilla chinensis extract in the feed played an optimizing role in the intestinal health and antioxidant capacity of Mastacembelus armatus.

[0178] II. Preparation of a Mastacembelus armatus feed containing Pulsatilla chinensis extract

[0179] (1) The formula of the Mastacembelus armatus feed containing Pulsatilla chinensis extract is as shown in Table 4:

[0180] Table 4 Formula of the Mastacembelus armatus feed containing Pulsatilla chinensis extract (g / kg)

[0181]

[0182] (2) The preparation methods of the modified starch and the feed are similar to those in "I. Preparation of a Mastacembelus armatus feed containing Pulsatilla chinensis".

[0183] Experimental Example VIII. Determination of the ability of Mastacembelus armatus to regulate and adapt to external temperature

[0184] 1. Experimental method:

[0185] The experimental Mastacembelus armatus fries were provided by a Mastacembelus armatus farm in Guangzhou, Guangdong Province. Before the experiment, the control feed (0 mg / kg) was fed first. After two weeks of temporary rearing, the Mastacembelus armatus fries with healthy physique and uniform size of 8 ± 2 cm were selected. They were randomly divided into 15 nylon nets with specifications of 1.5 m in length, 1.2 m in width and 1.0 m in height. Among them, every 3 nets were fed with the same kind of feed, that is, divided into 5 groups. The four corners of the four sides and the bottom were fixed with jars filled with sand, and a feed tray was hung in each net. A sunshade shed was built above the cultivation net cage to avoid direct sunlight. They were fed twice a day (at 9:00 and 17:00) until apparent satiety, for eight consecutive weeks.

[0186] The feeds fed to each group of Mastacembelus armatus were as follows:

[0187] Control Example 1 group: Fed with the feed prepared in Control Example 1;

[0188] Example 2 group: Fed with the feed prepared in Example 2;

[0189] Example 6 group: Fed with the feed prepared in Example 6;

[0190] Control Example 2 group: Fed with the feed prepared in Control Example 2; The preparation method of Control Example 2 was as follows: The difference from Example 6 was that the modified starch was replaced with commercially available pregelatinized starch (purchased from Shandong Dongchen Biotechnology Co., Ltd.);

[0191] Control Example 3 group: Fed with the feed prepared in Control Example 3; The preparation method of Control Example 3 was as follows: The difference from Example 6 was that 193.48 g / kg of the modified starch was replaced with 173.48 g / kg of commercially available pregelatinized starch (purchased from Shandong Dongchen Biotechnology Co., Ltd.), and 20 g / kg of sodium alginate was added.

[0192] After eight weeks of feeding, each group of Mastacembelus armatus was placed in an artificial climate incubator, treated at 28 °C (initial temperature group) for 24 h, then cooled to 20 °C (low temperature holding group) at a rate of 4 °C / h. After continuously treating at 20 °C for 24 h, it was then heated to 28 °C (recovery temperature group) at a rate of 4 °C / h and continuously treated for 24 h to observe the activities of the experimental Mastacembelus armatus. Sampling was carried out at the later stage of each temperature group treatment, and liver tissues were collected for the determination of antioxidant enzyme activity. The Catalase (CAT) was detected using the kit from Nanjing Jiancheng Bioengineering Research Institute Co., Ltd.

[0193] 2. Experimental results

[0194] The experimental results are shown in Table 5.

[0195] Table 5 Determination of the regulation and adaptation ability of Mastacembelus armatus to external temperature

[0196]

[0197] As can be seen from Table 5, the mixture of Pulsatilla chinensis extract and chitosan oligosaccharide used in a specific ratio can improve the ability of Mastacembelus armatus to regulate the external temperature, which is more conducive to the growth and reproduction of Mastacembelus armatus.

[0198] In addition, from November 1, 2023 to March 1, 2024, in a Mastacembelus armatus farm in Guangzhou City, Guangdong Province, the Mastacembelus armatus were fed with the Mastacembelus armatus feed containing Pulsatilla chinensis extract provided by the present invention. The overwintering survival rate of Mastacembelus armatus was 90%, which was at least 25% higher than the survival rate of using fresh animal feeds such as Tenebrio molitor, earthworms or maggots in previous years. This shows that using the Mastacembelus armatus feed containing Pulsatilla chinensis extract provided by the present invention is more conducive to the artificial breeding of Mastacembelus armatus.

[0199] The above embodiments are only illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. Any person familiar with this technology can modify or change the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or changes completed by those with ordinary knowledge in the technical field without departing from the spirit and technical idea disclosed by the present invention should still be covered by the claims of the present invention.

Claims

1. A Mastacembelus armatus feed containing Pulsatilla chinensis extract, characterized in that, It comprises the following components and their parts by weight: 42 - 46 parts of white fish meal, 15 - 20 parts of extruded soybean, 15 - 20 parts of modified starch, 1 - 3 parts of brewer's yeast, 6 - 8 parts of fish oil, 2 - 4 parts of soybean oil, 1 - 2 parts of soy lecithin, 0.4 - 0.6 part of choline chloride, 0.4 - 0.6 part of vitamin C phosphate, 1 - 2 parts of calcium dihydrogen phosphate, 0.4 - 0.6 part of L-alanine, 1 - 3 parts of vitamin and mineral premix, and 0.01 - 0.12 part of additive, wherein the additive is the extract of Pulsatilla chinensis.

2. The Mastacembelus armatus feed containing Pulsatilla chinensis extract according to claim 1, characterized in that, It comprises the following components and their parts by weight: 45 parts of white fish meal, 18 parts of extruded soybean, 15 - 20 parts of modified starch, 2 parts of brewer's yeast, 7 parts of fish oil, 3 parts of soybean oil, 1 part of soy lecithin, 0.5 part of choline chloride, 0.6 part of vitamin C phosphate, 1 part of calcium dihydrogen phosphate, 0.5 part of L-alanine, 2 parts of vitamin and mineral premix, and 0.03 part of additive, wherein the additive is the extract of Pulsatilla chinensis.

3. The Mastacembelus armatus feed containing Pulsatilla chinensis extract according to claim 1 or 2, characterized in that, The extract of Pulsatilla chinensis is the extract of the dried root of Pulsatilla chinensis.

4. The Mastacembelus armatus feed containing pulsatilla extract according to claim 1 or 2, characterized in that The preparation method of the modified starch is as follows: Step A: Mix choline chloride and mannose and stir evenly. The mass ratio of choline chloride to mannose is 2:1 to obtain a mixed solvent. Step B: Add corn starch into the mixed solvent prepared in Step S1 and stir evenly to obtain a starch suspension. Adjust the pH of the starch suspension to 7.5 - 8.5, gelatinize at 90 - 95 °C for 1 - 2 h. After the reaction, add absolute ethanol, filter by suction to obtain the precipitate, wash with absolute ethanol, and dry to obtain pregelatinized starch. Step C: Take sodium alginate and add it into distilled water and stir evenly to obtain a mixed solution. Add the pregelatinized starch prepared in Step B into the mixed solution, stir at 40 - 45 °C for 20 - 30 min, dry, pulverize, and sieve through a 100-mesh sieve to obtain the product.

5. The Mastacembelus armatus feed containing Pulsatilla chinensis extract according to claim 4, wherein, In Step B, the mass concentration of corn starch in the mixed solvent is 0.8 - 1.2 wt%.

6. The Mastacembelus armatus feed containing Pulsatilla chinensis extract according to claim 4, characterized in that, In Step C, the mass ratio of pregelatinized starch to sodium alginate is 1:(0.05 - 0.1).

7. The Mastacembelus armatus feed containing Pulsatilla chinensis extract according to claim 1 or 2, characterized in that The additive further comprises chitosan oligosaccharide.

8. The Mastacembelus armatus feed containing pulsatilla extract according to claim 7, wherein, In the additive, the mass ratio of the extract of Pulsatilla chinensis to chitosan oligosaccharide is 1:(0.4 - 0.8).

9. The Mastacembelus armatus feed containing Pulsatilla chinensis extract according to claim 8, characterized in that, In the additive, the mass ratio of the extract of Pulsatilla chinensis to chitosan oligosaccharide is 1:0.

6.

10. The preparation method of the Mastacembelus armatus feed containing Pulsatilla chinensis extract according to any one of claims 1 to 9, characterized in that, It comprises the following steps: Step S1: Take half of the modified starch and mix it evenly with vitamin and mineral premix, additive, vitamin C phosphate, fish oil, and soybean oil to obtain mixture I. Then add 35 - 40% of the total mass of the above mixture I of water and stir evenly to obtain mixture II. Step S2: Mix the remaining modified starch evenly with white fish meal, extruded soybean, brewer's yeast, soy lecithin, choline chloride, calcium dihydrogen phosphate, and L-alanine. Then add the mixture II prepared in Step S1, mix well to obtain mixture III. Then add 30% of the total mass of the above mixture III of water, make it into pellet feed, and dry in the air to obtain the product.

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