Feed additive for relieving diarrhea of weaned piglets and improving intestinal health and application of feed additive

Abstract

The invention discloses a feed additive for relieving diarrhea of weaned piglets and improving intestinal health and application of the feed additive. According to the invention, it is found for the first time that the growth performance of the weaned piglets can be improved and the diarrhea rate can be reduced by adding wormwood polysaccharides with different concentrations into daily ration of the weaned piglets; the apparent nutrient digestibility is improved, and the utilization rate of the piglets on nutrient substances such as protein, fat and mineral substances in the feed is promoted; the antioxidant capacity of serum is improved; the microbial flora structure is improved; the content of short-chain fatty acid is increased; the metabolic capability of intestinal microorganisms of piglets is improved. The application of the feed additive is expected to improve the efficiency and quality of piglet breeding, and the feed additive is more suitable for popularization and application in the breeding industry and has a wide application prospect. In addition, the invention provides a new solution thought and method for livestock production and animal growth.

Description

Technical Field

[0001] The invention belongs to the technical field of feed additives, and particularly relates to a feed additive for alleviating diarrhea in weaned piglets and improving intestinal health, and an application thereof. Background Art

[0002] As an important food production field, animal husbandry has a continuous demand for increasing the growth rate of animals, improving the breeding environment and improving the quality of meat. Pork is deeply favored by people and is one of the most popular meats. According to the data of the National Bureau of Statistics in 2023, the national pig output reached 726.62 million heads, pork production was 57.94 million tons, and the pig inventory was 43,422 heads. The pig farming industry is an important part of our people's livelihood, and weaning is one of the most critical periods in pig farming. The damage to the intestinal barrier function caused by early weaning of piglets can cause health problems such as slow growth and diarrhea, resulting in an increase in the mortality rate, which seriously affects the economic benefits of the farm and the output of pork. The use of a large amount of antibiotics and additives in feed in intensive farming not only affects the quality of pork, but also endangers human health and causes food safety problems. Against the backdrop of the current policy of banning antibiotics in feed and the trend towards ecological farming, the search for green and healthy natural products is particularly critical and urgent for alleviating weaning stress in piglets, improving growth performance, regulating intestinal barrier function, and reducing mortality. Feed products that can be applied to actual breeding production are of great significance.

[0003] At present, studies have found that polysaccharides from mugwort have good antioxidant effects, mainly by scavenging free radicals, resisting reactive oxygen species, and protecting the body from oxidative stress damage. In addition, polysaccharides from mugwort have certain enhancing effects on both nonspecific immunity and specific immunity, and can activate macrophages, enhance their phagocytic function and cytokine activity, and contribute to the secretion of nitric oxide (NO), thereby enhancing the body's immunity. Polysaccharides from mugwort can also inhibit the proliferation and differentiation of tumor cells. Studies have reported that the polysaccharide component (AAWAP) obtained from mugwort water extract can selectively inhibit platelet aggregation induced by podocalyx (PDPN), and inhibit platelet aggregation induced by PDPN and tumor cells by irreversibly blocking the interaction between PDPN and C-type lectin-like receptor2 (CLEC-2) signaling pathways in a dose-dependent manner. Furthermore, wormwood polysaccharides have a strong antibacterial effect. Some studies have reported the use of filter paper method to determine the antibacterial activity and minimum inhibitory concentration (MIC) of wormwood polysaccharides. The results showed that wormwood polysaccharides have a certain inhibitory effect on three common bacteria, and have a stronger inhibitory effect on Gram-positive bacteria such as Staphylococcus aureus and Bacillus subtilis.

[0004] Chinese herbal medicine has the advantages of small toxic side effects, no significant drug resistance, and abundant resources, and has important application potential in animal husbandry. Among them, Artemisia argyi Polysaccharides (AAP) is a heteropolysaccharide contained in wormwood. As a natural biomacromolecule, it has biological activities such as regulating intestinal flora, anti-oxidation, immunomodulation, anti-inflammatory, and anti-tumor. It has become a potential auxiliary treatment method in alleviating intestinal barrier damage and regulating intestinal health. At present, there are no reports on the use of AAP to relieve weaning stress, improve growth performance, regulate intestinal barrier function, and reduce mortality in piglets. Summary of the invention

[0005] In order to overcome the deficiencies of the above-mentioned prior art, the present invention proposes a feed additive for alleviating diarrhea in weaned piglets and improving intestinal health. The feed additive AAP is applied to the basic pig feed to effectively alleviate the weaning stress of piglets, improve the growth performance of weaned piglets, reduce the diarrhea rate, increase the apparent nutrient digestibility, improve the antioxidant capacity, improve the microbial flora structure, increase the short-chain fatty acid content, improve the metabolic capacity of the intestinal microorganisms of piglets, and promote the overall growth state of piglets in many aspects, and has important application prospects in piglet breeding.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is:

[0007] A first aspect of the present invention provides a feed additive for alleviating diarrhea in weaned piglets and improving intestinal health. The feed additive is AAP.

[0008] The second aspect of the present invention provides the use of the feed additive described in the first aspect in the preparation of a feed for alleviating diarrhea in weaned piglets and improving intestinal health. The feed additive is used to improve the growth performance of weaned piglets and reduce the diarrhea rate; or / and to improve the apparent nutrient digestibility; or / and to increase the activity of serum antioxidant enzymes and reduce the damage of oxygen free radicals to the body; or / and to improve the structure of intestinal microbial flora; or / and to increase the content of intestinal short-chain fatty acids.

[0009] Furthermore, the serum antioxidant enzymes mainly include total superoxide dismutase (T-SOD), glutathione peroxidase (GSH-Px) and catalase (CAT) as well as malondialdehyde (MDA), the main component of lipid peroxidation products.

[0010] Furthermore, the feed additive is used to improve the growth performance of weaned piglets and reduce the diarrhea rate. As the content of wormwood polysaccharide in the feed increases, the diarrhea index of the weaned piglets decreases in a quadratic curve, and adding 500 to 1500 mg / kg of the feed additive can improve their diarrhea condition.

[0011] Furthermore, the feed additive is used to improve the apparent nutrient digestibility of weaned piglets, and the addition of wormwood polysaccharides in the feed can promote the piglets' utilization of protein, fat, and mineral nutrients in the feed.

[0012] Furthermore, the feed additive is used to improve the intestinal microbial flora structure mainly including phylum level, genus level, Alpha diversity, Beta diversity analysis, LEfSe species difference analysis, and increase the content of intestinal short-chain fatty acids.

[0013] Compared with the prior art, the present invention has the following beneficial effects:

[0014] AAP in the present invention can improve the structure of intestinal flora and increase the concentration of short-chain fatty acids of microbial metabolites, thereby effectively alleviating diarrhea symptoms of piglets and promoting their growth. At the same time, AAP can promote the utilization rate of nutrients such as protein, fat, and minerals in feed by piglets, and reduce the feed-to-weight ratio of weaned piglets to a certain extent. AAP can increase the activity of GSH-Px and T-AOC in piglet serum, enhance the antioxidant capacity of weaned piglets, and thus reduce the damage of oxygen free radicals to the body. Therefore, the feed of the present invention is expected to improve the efficiency and quality of piglet breeding, and is more suitable for promotion and application in animal husbandry, and has broad application prospects. In addition, the present invention provides new solutions and methods for animal husbandry production and animal health. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Analysis of rectal microbial composition. (A) The top 10 species in relative abundance at the phylum level and analysis. (B) The top 10 species in relative abundance at the genus level and analysis. (C) Species with significant differences at the genus level. (D) Species with significant differences at the genus level.

[0016] Figure 2 This is the rectal dilution curve.

[0017] Figure 3 (A) NMDS analysis; (B) UPGMA analysis for the impact of rectal fecal community β diversity.

[0018] Figure 4 This is the difference analysis of LEfSe species (LDA greater than 3): (A) Differential microbial flora analysis between 0 mg / kg group and 1000 mg / kg group; (B) Differential microbial flora analysis between 0 mg / kg group and 2000 mg / kg group; (C) Differential microbial flora analysis between 1000 mg / kg group and 2000 mg / kg group. DETAILED DESCRIPTION

[0019] The specific embodiments of the present invention are further described below. It should be noted that the description of these embodiments is used to help understand the present invention, but does not constitute a limitation of the present invention. In addition, the technical features involved in each embodiment of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0020] The experimental methods in the following examples are conventional methods unless otherwise specified, and the experimental materials used in the following examples are commercially available unless otherwise specified.

[0021] General Example: A feed additive for reducing diarrhea rate of weaned piglets, increasing antioxidant capacity and improving intestinal flora structure and its application

[0022] Example 1: Effect of adding 500 mg / kg AAP to feed on diarrhea, antioxidant capacity and intestinal flora of weaned piglets. Example 2: Effect of adding 1000 mg / kg AAP to feed on diarrhea, antioxidant capacity and intestinal flora of weaned piglets. Example 3: Effect of adding 1500 mg / kg AAP to feed on diarrhea, antioxidant capacity and intestinal flora of weaned piglets. Example 4: Effect of adding 2000 mg / kg AAP to feed on diarrhea, antioxidant capacity and intestinal flora of weaned piglets.

[0023] 1. Materials and Methods

[0024] 1.1 Experimental feed

[0025] According to the nutritional needs of piglets, five groups of isonitrogen and isoenergetic feeds were designed. The feed formula is shown in Table 1. The main sources of protein are soybean meal and fish meal, the main source of fat is soybean, and the main source of carbohydrates is puffed corn and broken rice. The control group was fed with a basic feed, and the other treatment groups added 500, 1000, 1500 and 2000 mg / kg of wormwood polysaccharides to the basic feed. Among them, wormwood polysaccharides (AAP) were purchased from Shaanxi Jinrun Biotechnology Co., Ltd., with a polysaccharide content of 90%. DSS (dextran sulfate sodium) was purchased from MP Company in the United States, with a molecular weight of 36-50 kDa, and was added to the basic feed of piglets by direct addition and stirring.

[0026] Table 1 Basic diet composition and nutritional level

[0027]

[0028]

[0029] Note: Each kilogram of basic daily diet contains: crude protein ≥18.0%, crude fiber ≤4.0%, crude ash ≤8.0%, calcium 0.5%~1.2%, lysine ≥1.3%, sodium chloride 0.2%~0.8%, total phosphorus ≥0.5%, and moisture ≤14%.

[0030] 1.2 Experimental design and breeding management

[0031] Experimental location: Xinxiang Lvfengyuan Agriculture Co., Ltd. (Xinxiang City, Henan Province).

[0032] Experimental animals: 35 28-day-old weaned piglets (Du×Chang×Da) were purchased from Xinxiang Lvfengyuan Agriculture Co., Ltd. (Xinxiang City, Henan Province).

[0033] A single-factor randomized group design was used to select 35 28-day-old, healthy, average-weight (7.30±0.04kg) three-way crossbred (Durham × Long × Long) weaned piglets and randomly divided them into 5 groups, with 7 replicates in each group and 1 pig in each replicate. The control group was fed with a basic feed, and the other treatment groups were supplemented with 500, 1000, 1500 and 2000 mg / kg of AAP, respectively, on the basic feed. The experimental period was 14 days.

[0034] Before the start of the experiment, the weaned piglet breeding environment was disinfected, and the feed troughs and waterers were cleaned. After the start of the experiment, the feces in the pen corridors and pens were cleaned every morning. During the experiment, weaned piglets were free to eat and ensured to have sufficient drinking water. Weaned piglets were fed 5 times a day (feeding time was 07:00, 10:00, 14:00, 17:00 and 19:00), and the amount of each addition was appropriate to have a little surplus in the trough. The mental state of the weaned piglets was observed at 8:00 and 17:00 every day, and piglets with poor mental state and loss of appetite were observed, and diarrhea of ​​the piglets was recorded. During the experiment, the temperature of the pig house was maintained at 25℃~28℃, and the relative humidity was 60%~70%. The pigs were weighed on an empty stomach at 8:00 am on the 1st and 15th days of the experiment. The daily management of the experimental site and the immunization measures of weaned piglets were carried out in accordance with the standard production procedures of the pig farm.

[0035] 1.3 Sample collection

[0036] Feed samples: Add Cr2O3 indicator to the feed, ensure uniform mixing, and then use multi-point sampling method to collect about 200g of feed samples and store them at -20℃ for subsequent use.

[0037] Fecal samples: On the 7th day of the feeding experiment, each group was subjected to a digestion test with each replicate (per pen) as the unit. 0.25% Cr2O3 (analytical grade) was evenly mixed into the diet. The pre-feeding period was 2 days, and the formal feces collection period was 5 days. The piglet feces were collected at 8:00 am and 15:00 pm every day for 5 consecutive days. 10wt% sulfuric acid was added to the collected feces to fix nitrogen, and then stored at -20℃.

[0038] Serum samples: After the experiment, all weaned piglets were fasted for 24 hours. After weighing, 5 mL of blood was collected from the anterior vena cava using a 5 mL disposable syringe, transferred to a 10 mL centrifuge tube (do not shake to prevent hemolysis), left to stand at room temperature for 30 min to 2 h, centrifuged at 3000 g / min for 15 min, and the collected serum was divided into 1.5 mL centrifuge tubes and stored at -80 °C for testing.

[0039] Rectal fecal microbial samples: On the morning of the 15th day of the experiment, two samples of rectal feces were collected by massaging the anus of weaned piglets and stored at -80°C.

[0040] 1.4 Test methods

[0041] (1) Growth performance

[0042] After fasting for 12 hours, the experimental pigs were weighed on an empty stomach in the morning of the 1st and 15th days of the experiment, and the initial and final weights of the piglets in each group were recorded to calculate ADG (average daily weight gain); the daily feed intake was recorded during the entire experimental period, and ADFI (average daily feed intake) was calculated; the feed-to-weight ratio (F / G) was calculated based on ADFI and ADG.

[0043] Stool scores and diarrhea rates

[0044] The incidence and severity of piglet diarrhea were evaluated by fecal consistency scoring at 8:00 a.m. and 17:00 p.m. every day. The fecal condition of each piglet was observed and recorded. The criteria for judging piglet diarrhea are shown in Table 2.

[0045] Diarrhea index = total diarrhea scores of piglets in each group during the test period / (number of test days × number of piglets in each group)

[0046] Table 2 Stool scoring criteria

[0047]

[0048] (2) Determination of nutrient apparent digestibility

[0049] The determination of DM, CP, EE, NDF, ADF, Ash, Ca, P and other indicators in feed and feces was carried out according to Zhang Liying (2003). The content of Cr2O3 in feed and feces was determined by atomic absorption spectrophotometer. The pretreatment of Cr2O3 in feed and feces was as follows:

[0050] Feed and feces samples were dried at 65°C and crushed, and about 0.3g of uniform dry sample was weighed to an accuracy of 0.0001g. In a 100mL conical flask, 10mL of mixed acid (volume ratio: concentrated nitric acid: perchloric acid = 4:1) was added, and a bent-neck funnel was covered. It was left to stand overnight, and then placed on a hot plate in a fume hood and digested with a low heat until white smoke appeared and the digestion liquid was clear. (If the digestion liquid has black carbon particles or is brown-black, a few mL of mixed acid can be added and the digestion can be continued by heating until the digestion liquid is clear). Because the amount of acid is high, it is necessary to continue to heat with a high heat to drive the acid to about 1-2mL. After cooling, it is transferred to a 100mL volumetric flask with a 2wt% nitric acid solution and mixed for use. A blank test was performed at the same time.

[0051] The calculation formula for the apparent digestibility of each nutrient is: apparent digestibility of a certain nutrient (%) = (1-A2 / A1×B1 / B2)×100%; A1 is the content of a certain component in the feed (%), A2 is the content of the corresponding component in the feces (%), B1 is the content of Cr2O3 in the feed (%), and B2 is the content of Cr2O3 in the feces (%).

[0052] (3) Determination of serum antioxidant enzyme activity

[0053] The antioxidant enzyme activities of T-AOC (total antioxidant capacity), T-SOD (total superoxide dismutase), GSH-Px (glutathione peroxidase), and MDA (malondialdehyde) in serum samples were determined by microplate reader. The detection kit used in this experiment was purchased from Nanjing Jiancheng Bioengineering Institute, and its operation process was strictly carried out in accordance with the kit instructions.

[0054] (4) Analysis of rectal fecal microbial composition

[0055] Three groups of 0, 1000, and 2000 mg / kg AAP were selected for rectal microbial composition analysis, and rectal content samples were sent to Tianjin Novogene Bioinformatics Technology Co., Ltd. for 16S rDNA sequencing. The specific analysis process included the following operations. Genomic DNA extraction and sample detection: Total bacterial DNA was extracted using a DNA kit, and DNA concentration and integrity were detected by Agilent 5400. PCR amplification: This experiment used primers in the V3-V4 hypervariable region of the 16S rDNA gene for PCR amplification. The primer sequences are as follows:

[0056] 515F:5'-CCTAYGGGRBGCASCAG-3'

[0057] 806R:5'-GGACTACNNNGGGTATCTAAT-3'

[0058] Purification of PCR products: PCR products were detected by electrophoresis using 2 wt% agarose gel; qualified PCR products were purified by magnetic beads.

[0059] Library construction and upstream sequencing: A small fragment library was constructed according to the characteristics of the amplified region. The constructed library was quantified by Qubit and Q-PCR. After the library was qualified, PE250 sequencing was performed using NovaSeq6000.

[0060] Sequencing data processing: The raw data of each sample was obtained by splitting according to the barcode, and the barcode and primer were removed. Then, the R1 and R2 sequence data were spliced ​​by FLASH software. The spliced ​​tags were quality controlled to obtain Clean Tags, and then chimeras were filtered to obtain effective data (Effective Tags) that can be used for subsequent analysis. Then, DADA2 was used to reduce noise and duplicate, and the sequences were clustered into ASVs with 100% similarity, and representative ASVs were annotated for species. Alpha diversity index (ACE, chao1, goods_coverage, observed_species, shannon, simpson) was calculated based on the annotation results of ASVs to analyze the diversity of microbial communities in the samples. According to species annotation and ASV abundance data, similar ASV information was merged to form a species abundance table. Beta diversity analysis was further performed using the ASV phylogenetic relationship to explore the differences in microbial community structure in different groups. PCoA analysis was performed to display the differences in community structure composition in two dimensions. LEfSe analysis was used to find biomarkers with statistical differences between different groups.

[0061] (5) Analysis of short-chain fatty acids

[0062] The rectal fecal samples were thawed and mixed. 1 g of the sample was placed in a 10 mL centrifuge tube, and 5 mL of deionized water (ddH2O) was added. After thoroughly mixing for 30 min using a vortex mixer, it was centrifuged at 15000 g / min at 4 °C for 15 min. Subsequently, 3.6 mL of the supernatant was taken and mixed with 0.4 mL of 25 wt% metaphosphoric acid solution, and fixed overnight. On the second day, the mixed solution was centrifuged at 15000 g / min for 10 min, then 2 mL of the supernatant was taken and filtered through a microporous membrane with a pore size of 0.45 μm. The filtered sample was detected using gas chromatography (Agilent Technologies 7890B GC System, Agilent, USA). The gas chromatography conditions were as follows: the chromatographic column was a DB-FFAP column (30 m × 250 μm × 0.25 μm); the carrier gas was high-purity nitrogen, and the flow rate was set at 0.8 mL / min. The temperature of the chromatographic column, the temperature of the flame ionization detector (FID), the detector temperature, and the injector temperature were set at 220 °C, 280 °C, and 250 °C, respectively.

[0063] 1.5 Data analysis

[0064] The experimental data were statistically analyzed using SPSS 22.0 software. For data of different groups, one-way analysis of variance (ANOVA) was used, and the effects of adding different concentrations of Artemisia argyi polysaccharide in the feed on various indicators of weaned piglets were determined through linear and quadratic effects, and multiple comparisons were combined with the Duncan method. All data were presented in the form of mean and standard error of the mean (SEM). P < 0.05 indicated a significant difference, and 0.05 < P < 0.10 indicated a trend of difference. GraphPad Prism 8.0.2 and Adobe Illustrator 2023 software were used for plotting.

[0065] 2. Experimental results

[0066] 2.1 Effects of adding Artemisia argyi polysaccharide in the feed on the growth performance and diarrhea rate of weaned piglets

[0067] The effects of adding Artemisia argyi polysaccharide in the feed on the growth performance and diarrhea rate of weaned piglets are shown in Table 3. As can be seen from Table 3, compared with the control group, the 500 mg / kg AAP group and the 1500 mg / kg AAP group significantly increased the ADG of weaned piglets (P = 0.050); as the AAP level in the feed increased, the diarrhea index showed a quadratic curve effect change (quadratic, P = 0.010). There were no significant differences in ADFI and F:G among different concentrations of AAP (P > 0.05). As the AAP content in the feed increased, the diarrhea index of weaned piglets showed a quadratic curve decreasing effect, indicating that 500 - 1500 mg / kg AAP could improve their diarrhea condition.

[0068] Table 3 Effects of Artemisia argyi polysaccharides on growth performance and diarrhea rate of weaned piglets

[0069]

[0070] Note: In the same data, different lowercase letters with superscripts indicate significant differences (P<0.05), while no superscript or the same lowercase letters with superscripts indicate no significant differences (P>0.05).

[0071] 2.2 Effect of adding wormwood polysaccharides to feed on apparent nutrient digestibility in weaned piglets

[0072] The effects of wormwood polysaccharides on the apparent nutrient digestibility of weaned piglets are shown in Table 4. The results showed that compared with the 1000 mg / kg and 2000 mg / kg AAP groups, the activity of T-SOD in the serum of weaned piglets in the 1500 mg / kg AAP group tended to decrease (P=0.073). As the level of AAP in the feed increased, the activity of GSH-Px in weaned piglets increased linearly (linear, P=0.006), and it showed a quadratic curve effect change (quadratic, P=0.045). In addition, as the level of AAP in the feed increased, the activity of T-AOC in the serum of weaned piglets increased linearly (linear, P=0.009). There was no significant difference in the content of MDA in weaned piglets in different groups (P>0.05). As the AAP level in the feed increased, the apparent digestibility of EE in weaned piglets showed a quadratic curve effect, and the apparent digestibility of Ca was linearly increased; compared with the 0, 1000, and 1500 mg / kg AAP groups, the apparent digestibility of CP in weaned piglets in the 500 mg / kg AAP group showed a significant increase. In addition, the 500 mg / kg AAP group significantly increased the apparent digestibility of Ash in weaned piglets. These results indicate that the addition of AAP in feed can promote the utilization of nutrients such as protein, fat, and minerals in feed by piglets, further explaining that the addition of AAP can reduce the feed-to-weight ratio of weaned piglets to a certain extent.

[0073] Table 4 Effect of AAP on apparent digestibility of nutrients in weaned piglets

[0074]

[0075]

[0076] 2.3 Effect of Adding Artemisia annua Polysaccharides to Feed on Serum Antioxidant Capacity of Weaned Piglets

[0077] Effect of Artemisia argyi polysaccharide on the antioxidant capacity of serum of weaned piglets Table 5 shows that compared with the 1000mg / kg and 2000mg / kg AAP groups, the activity of T-SOD in the serum of weaned piglets in the 1500mg / kg AAP group tended to decrease (P=0.073). With the increase of AAP level in the feed, the activity of GSH-Px in weaned piglets was linearly increased (linear, P=0.006), and it showed a quadratic curve effect change (quadratic, P=0.045). In addition, with the increase of AAP level in the feed, the activity of T-AOC in the serum of weaned piglets was linearly increased (linear, P=0.009). There was no significant difference in the content of MDA in weaned piglets in different groups (P>0.05). Adding AAP to the feed of weaned piglets can increase the activity of GSH-Px and T-AOC in serum and reduce the content of MDA to a certain extent. This shows that AAP can inhibit the increase of MDA content, while increasing the activity of antioxidant enzymes, accelerating the removal of free radicals in the body, and enhancing the antioxidant capacity of weaned piglets, thereby reducing the damage of oxygen free radicals to the body.

[0078] Table 5 Effect of AAP on serum antioxidant capacity of weaned piglets

[0079]

[0080] 2.4 Effect of adding mugwort polysaccharides to feed on the microbial composition of rectal feces in weaned piglets

[0081] 2.4.1 Analysis of bacterial flora composition

[0082] Effect of Artemisia argyi polysaccharide on the microbiota composition of weaned piglets Figure 1 As shown, Figure 1 (A) At the phylum level, the microbial flora of the three groups was mainly composed of Firmicutes and Bacteroidota, and the sum of their relative abundances exceeded 94%. At the same time, the relative abundance of Firmicutes in the 1000mg / kg and 2000mg / kg AAP groups was higher than that in the 0mg / kg AAP group, while the relative abundance of Bacteroidota was lower than that in the 0mg / kg AAP group. Figure 1 In (B), we can see that at the genus level, the three groups of microbial flora are mainly composed of Prevotella_9, Megasphaera, Phascolarctobacterium, Blautia, Roseburia, [Ruminococcus]_torques_group, Lactobacillus, Alloprevotella, Holdemanella, and Rikenellaceae_RC9_gut_group. Figure 1As shown in (B / C), with the increase of AAP level in the feed, the relative abundance of Alloprevotella decreased linearly (linear, P = 0.039), while the relative abundance of [Ruminococcus]_torques_group (quadratic, P = 0.068) tended to increase.

[0083] 2.4.2 Alpha Diversity Analysis

[0084] like Figure 2 As shown in Table 6, the sequencing depth index of the three groups with 0, 1000 mg / kg, and 2000 mg / kg AAP was as high as 100%; and the dilution curves of the three groups tended to be flat, indicating that the amount of sequencing data was reasonable, and the sequencing depth had basically covered all species in the sample, which was sufficient to reflect the species diversity in the sample. There was no significant difference in the Alpha diversity index of rectal microorganisms of weaned piglets in different groups (P>0.05).

[0085] Table 6 Effect of AAP on α-diversity of rectal fecal community in weaned piglets

[0086]

[0087] 2.4.3 Beta diversity analysis

[0088] Depend on Figure 3 As shown in (A), the non-metric multi-dimensional scaling (NMDS) analysis showed that there was no obvious difference between the three groups of rectal fecal microbial samples. The UPGMA (Unweighted Pair-group Method with Arithmetic Mean) cluster analysis showed that the three groups of rectal fecal microbial samples were not significantly different. Figure 3 As shown in (B), the rectal fecal microbial flora structure of the 1000 mg / kg AAP group and the 2000 mg / kg AAP group was more similar than that of the 0 mg / kg AAP group.

[0089] 2.4.4 Species Difference Analysis of LEfSe

[0090] Through LEFSE analysis, the biomarkers with significant enrichment in the rectal microbial composition of three groups of weaned piglets with different treatments were screened out (the LDA threshold was 3). Figure 3-4In (A), compared with the 0mg / kgAAP group, the significantly enriched Biomarkers in the 1000mg / kg AAP group were Selenomonadaceae family, Agathobacter genus, Butyricimonas genus, Marinifilaceae family, UCG_002 genus, RF39 order, Archaea kingdom, Euryarchaeota phylum, Methanobacteria class, Methanobrevibacter genus, Methanobacteriaceae family, Methanobacteriales order, Eubacterium__ruminantium_group genus, and Fusicatenibacter genus. The significantly enriched Biomarkers in the 0mg / kgAAP group were Bacteria kingdom, Lactobacillus_mucosae species, Ligilactobacillus genus, and Limosilactobacillus genus. Figure 3-4 In (B), compared with the 0mg / kgAAP group, the significantly enriched Biomarkers in the 2000mg / kgAAP group were Selenomonadaceae family, Oscillospiraceae family, Treponema genus, Peptococcaceae family, Peptococcales order, UCG_004 species, Treponema_berlinense genus, Oscillibacter genus, and Eggerthellaceae family. The significantly enriched Biomarkers in the 0mg / kg AAP group were Alloprevotella genus, Roseburia_intestinalis species, Ligilactobacillus genus, Prevotella_sp_DJF_LS16 species, GCA_900066575 genus, Limosilactobacillus genus, and Lactobacillus_mucosae species. Figure 3-4 In (C), compared with the 1000 mg / kg AAP group, the significantly enriched biomarkers in the 2000 mg / kg AAP group were Intestinimonas, Oscillospiraceae, and Colidextribacter. The significantly enriched biomarkers in the 1000 mg / kg AAP group were Lachnospiraceae, Lachnospirales, Enterobacteriaceae, and Prevotella_sp_DJF_LS16.

[0091] 2.5 Effect of adding mugwort polysaccharides to feed on the concentration of short-chain fatty acids (SCFAs) in rectal feces

[0092] As shown in Table 7, compared with the 500mg / kg, 1500mg / kg, and 2000mg / kg AAP groups, the content of acetic acid (P < 0.001) in the 1000mg / kg AAP group was significantly increased; compared with the 0 and 1500mg / kg AAP groups, the content of butyric acid (P < 0.001) and valeric acid (P = 0.001) in the 1000mg / kg AAP group was significantly increased. In addition, the content of propionic acid (P = 0.015) in the 1000mg / kg AAP group was significantly higher than that in the other groups. With the increase of AAP levels in the feed of weaned piglets, the content of isobutyric acid (quadratic, P = 0.051) showed a trend of quadratic curve effect. There was no significant difference in the content of isovaleric acid in the rectal feces of weaned piglets in different groups (P > 0.05). The study found that the 1000mg / kg AAP group can significantly increase the concentrations of propionic acid, butyric acid, and valeric acid in the rectal feces of piglets. The increase in SCFAs content in the experiment shows that the addition of AAP has a positive regulatory effect on the intestinal flora of piglets, and its action pathway may be through increasing the abundance of SCFAs-producing bacteria and improving the metabolic capacity of intestinal microorganisms in piglets. Therefore, AAP promotes the production of SCFAs by regulating the abundance and metabolic process of microbial genera, while inhibiting the growth and colonization of harmful bacteria, creating a good intestinal environment, improving the digestion and absorption of nutrients by piglets, and enhancing their ability to resist oxidative stress, thereby alleviating the stress of weaned piglets.

[0093] Table 7 Effect of AAP on short-chain fatty acids in rectal feces of weaned piglets

[0094]

[0095]

[0096] In summary, adding 500-1000 mg / kg AAP to the diet can change the structure of intestinal flora, promote the production of intestinal flora metabolites SCFAs, thereby improving intestinal barrier function, enhancing antioxidant stress resistance, and alleviating weaning stress in piglets, thereby improving piglet diarrhea and improving their growth performance.

[0097] The embodiments of the present invention are described in detail above, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions and variations of these embodiments are made without departing from the principles and spirit of the present invention, and still fall within the protection scope of the present invention.

Claims

1. A feed additive for alleviating diarrhea in weaned piglets and improving intestinal health, characterized in that: The feed additive is wormwood polysaccharide.

2. Use of the feed additive according to claim 1 in preparing feed for alleviating diarrhea in weaned piglets and improving intestinal health.

3. The use according to claim 2, characterized in that: The feed additive is added in an amount of 500 to 2000 mg / kg in the feed.

4. The use according to claim 2, characterized in that: The feed additive is used to improve the growth performance of weaned piglets and reduce the diarrhea rate; or / and to improve the apparent nutrient digestibility; or / and to improve the serum antioxidant enzyme activity to reduce the damage of oxygen free radicals to the body; or / and to improve the intestinal microbial flora structure; or / and to increase the intestinal short-chain fatty acid content.

5. The use according to claim 4, characterized in that: The serum antioxidant enzymes mainly include total superoxide dismutase (T-SOD), glutathione peroxidase (GSH-Px) and catalase (CAT) as well as malondialdehyde (MDA), a main component of lipid peroxidation products.

6. The use according to claim 2, characterized in that: The feed additive is used to improve the growth performance of weaned piglets and reduce the diarrhea rate. As the content of wormwood polysaccharide in the feed increases, the diarrhea index of the weaned piglets decreases in a quadratic curve. Adding 500 to 1500 mg / kg of the feed additive can improve the diarrhea condition of the piglets.

7. The use according to claim 2, characterized in that: The feed additive is used to improve the apparent nutrient digestibility of weaned piglets, and the addition of wormwood polysaccharide in the feed can promote the piglets' utilization of protein, fat and mineral nutrients in the feed.

8. The use according to claim 2, characterized in that: The feed additive is used to improve the intestinal microbial flora structure, mainly including phylum level, genus level, Alpha diversity, Beta diversity analysis, LEfSe species difference analysis, and increase the content of intestinal short-chain fatty acids.

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