Application of Chinese yam in preparation of feed for improving immunity of weaned piglets, enhancing oxidation resistance of weaned piglets and improving integrity of intestinal barrier

Abstract

The invention provides an application of Chinese yam in preparation of a feed for improving the immunity of weaned piglets, enhancing the oxidation resistance of the weaned piglets and improving the integrity of intestinal barriers, and belongs to the technical field of livestock breeding. The Chinese yam is added into the feed, so that the body immunity and the disease resistance of weaned piglets can be improved; intestinal mucosa immunity is enhanced by promoting expression of intestinal defensin and interferon genes of weaned piglets; the intestinal barrier integrity and intestinal functions of weaned piglets can be improved; by activating a Keap1 / Nrf2 / HO-1 classical signal channel and improving antioxidant enzyme expression, the intestinal antioxidant capacity of the piglets is enhanced; and excessive apoptosis of intestinal mucosa epithelial cells caused by weaning of piglets can be effectively inhibited.

Description

Technical Field

[0001] The invention belongs to the technical field of animal husbandry, and in particular relates to an application of yam in preparing a feed for improving the immunity of weaned piglets, enhancing their antioxidant capacity and improving the integrity of the intestinal barrier. Background Art

[0002] With the development and expansion of the pig farming industry, early weaning of piglets is usually used to improve the production efficiency of the pig farming industry. Piglets are generally weaned 3-4 weeks after birth. Due to separation from sows and solid feed instead of breast milk, they are forced to adapt to new nutritional and environmental changes. In addition, their digestive function is not fully developed and their immune system is not perfect, which makes them prone to "weaning stress syndrome". This eventually leads to decreased feed intake, diarrhea, impaired intestinal immune function and digestion and absorption function of piglets, and they cannot fully digest, absorb and utilize these nutrients, thus providing a good source of nutrition for the reproduction of pathogens, thereby changing the composition of intestinal flora and causing intestinal flora imbalance. Intestinal microbial imbalance is also one of the important factors causing intestinal inflammation and diarrhea in newborns and post-weaning animals. Therefore, it is crucial to reduce the stress faced by young animals during weaning to ensure the stability of their intestinal environment.

[0003] In the past, antibiotics were often used in the livestock and poultry industry to relieve piglet weaning stress and improve livestock and poultry growth performance, but long-term use can lead to increased bacterial resistance, drug residues and environmental pollution, affecting both environmental and human health. As people pay more attention to food safety, the use of natural drugs for nutritional regulation in the early weaning period to improve the intestinal health of weaned piglets has received increasing attention in the breeding industry.

[0004] In recent years, single or compound plants have been gradually used as feed additives. For example, mulberry leaf powder is used to reduce the antioxidant capacity of intestinal serum in fattening pigs, and Chinese medicine residues composed of Prunella vulgaris, mulberry leaves and chamomile improve the growth performance, intestinal health and intestinal flora composition of weaned piglets. As a raw material for Chinese medicine additives, yam has good palatability and has both medicinal and edible value. Yam is the dried rhizome of Dioscorea zingiberensis, a plant of the Dioscoreaceae family. It is not only a traditional Chinese medicine, but also a common food. In recent years, it has attracted much attention due to its medicinal and nutritional value. The active ingredients of yam mainly include polysaccharides, mucins, phenols, alkaloids, etc., which have pharmacological effects such as immunomodulation, antioxidant, anti-inflammatory, anti-cancer, and regulation of blood sugar and blood lipids. However, the potential role of yam in the intestinal health of weaned piglets has not been reported. Summary of the invention

[0005] In view of this, the object of the present invention is to provide an application of yam in preparing feed for improving the immunity of weaned piglets, enhancing their antioxidant capacity and improving the integrity of the intestinal barrier.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The invention provides application of yam in preparing feed for improving intestinal health of weaned piglets.

[0008] The invention also provides the use of yam in preparing feed for improving the immunity of weaned piglets.

[0009] The present invention also provides the use of yam in preparing feed for improving the intestinal barrier integrity and intestinal function of weaned piglets.

[0010] The invention also provides the use of yam in preparing feed for improving the antioxidant capacity of weaned piglets.

[0011] The present invention also provides the use of yam in preparing feed for improving the titer of serum antibodies of weaned piglets, wherein the antibodies include CSFV-Ab and PRV-Ab.

[0012] The present invention also provides the use of yam in preparing feed for improving the gene expression level of intestinal defensins and interferons in weaned piglets. The defensins include pBD1 and pBD2, and the interferons include IFN-α, IFN-β and IFN-γ.

[0013] The present invention also provides the use of yam in preparing feed for improving the gene expression levels of Claudin-1, Occudin-1 and ZO-1 in the intestine of weaned piglets.

[0014] The present invention also provides the use of yam in preparing feed for regulating the Keap1 / Nrf2 / HO-1 antioxidant pathway in the intestine of weaned piglets, wherein the expression levels of Nrf2 and HO-1 are increased, and the expression level of Keap1 is decreased; the Keap1 / Nrf2 / HO-1 antioxidant pathway increases the expression level of antioxidant enzymes and decreases the expression level of oxidative damage factors, the antioxidant enzymes include Nqo1, CAT, SOD2, and the oxidative damage factors include Nos2.

[0015] The present invention also provides the use of yam in preparing feed for inhibiting intestinal cell apoptosis of weaned piglets. The apoptosis-related proteins include pro-apoptotic protein Bax and anti-apoptotic protein Bcl-2. The expression level of the pro-apoptotic protein Bax is reduced, and the expression level of the anti-apoptotic protein Bcl-2 is increased; the expression level of the anti-apoptotic protein Bcl-2 is increased to inhibit the expression of Cleaved-caspase-3, and the expression level of the Cleaved-caspase-3 protein is reduced.

[0016] Preferably, the added amount of the yam is 1-2%.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The invention adds yam to the feed to improve the immunity and disease resistance of weaned piglets; enhances intestinal mucosal immunity by promoting the expression of intestinal defensins and interferon genes in weaned piglets; improves the intestinal barrier integrity and intestinal function in weaned piglets; enhances the intestinal antioxidant capacity of piglets by activating the Keap1 / Nrf2 / HO-1 classical signaling pathway and increasing the expression of antioxidant enzymes; and effectively inhibits the excessive apoptosis of intestinal mucosal epithelial cells caused by weaning of piglets. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 is the effect of yam on antibody titer of weaned piglets (where A is the content of CSFV-Ab in the blood, B is the content of PRV-Ab in the blood, compared with the Control group *P<0.05, **P<0.01, ***P<0.001, all data are Mean±SEM, the same below);

[0020] Figure 2 The effect of yam on intestinal defensins and interferons in weaned piglets (A is the duodenum, B is the ileum, and C is the jejunum);

[0021] Figure 3 is the effect of yam on the intestinal barrier of weaned piglets (n=3) (wherein, A is the effect of yam on the expression level of tight junction protein in the intestinal barrier of weaned piglets; B is the effect of yam on the expression level of tight junction protein gene in the intestinal barrier of weaned piglets);

[0022] Figure 4 is the effect of yam on the Keap1 / Nrf2 / HO-1 antioxidant pathway in the intestine of weaned piglets (n=3) (where A is the effect of yam on the expression level of key antioxidant proteins in the intestine of weaned piglets; B is the effect of yam on the expression of antioxidant indicator genes in the intestine of weaned piglets);

[0023] Figure 5 The figure shows the effect of yam on intestinal apoptosis indexes of weaned piglets (n=3) (wherein, A shows the effect of yam on the expression level of intestinal apoptosis-related proteins of weaned piglets; B shows the expression level of Bax protein in duodenum, jejunum and ileum detected by immunohistochemistry (200× scale bar 50μm). DETAILED DESCRIPTION

[0024] The invention provides application of yam in preparing feed for improving intestinal health of weaned piglets.

[0025] The invention also provides the use of yam in preparing feed for improving the immunity of weaned piglets.

[0026] The present invention also provides the use of yam in preparing feed for improving the intestinal barrier integrity and intestinal function of weaned piglets.

[0027] The invention also provides the use of yam in preparing feed for improving the antioxidant capacity of weaned piglets.

[0028] The present invention also provides the use of yam in preparing feed for improving the titer of serum antibodies of weaned piglets, wherein the antibodies include CSFV-Ab and PRV-Ab.

[0029] The present invention also provides the use of yam in preparing feed for improving the gene expression level of intestinal defensins and interferons in weaned piglets. The defensins include pBD1 and pBD2, and the interferons include IFN-α, IFN-β and IFN-γ.

[0030] The present invention also provides the use of yam in preparing feed for improving the gene expression levels of Claudin-1, Occudin-1 and ZO-1 in the intestine of weaned piglets.

[0031] The present invention also provides the use of yam in preparing feed for regulating the Keap1 / Nrf2 / HO-1 antioxidant pathway in the intestine of weaned piglets, wherein the expression levels of Nrf2 and HO-1 are increased, and the expression level of Keap1 is decreased; the Keap1 / Nrf2 / HO-1 antioxidant pathway increases the expression level of antioxidant enzymes and decreases the expression level of oxidative damage factors, the antioxidant enzymes include Nqo1, CAT, SOD2, and the oxidative damage factors include Nos2.

[0032] The present invention also provides the use of yam in preparing feed for inhibiting intestinal cell apoptosis of weaned piglets. The apoptosis-related proteins include pro-apoptotic protein Bax and anti-apoptotic protein Bcl-2. The expression level of the pro-apoptotic protein Bax is reduced, and the expression level of the anti-apoptotic protein Bcl-2 is increased; the increased expression level of the anti-apoptotic protein Bcl-2 inhibits the expression of Cleaved-caspase-3, and the expression level of the Cleaved-caspase-3 protein is reduced.

[0033] In the present invention, the added amount of the yam is 1-2%.

[0034] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0035] Materials used in the present invention: ZO-1, Occludin-1, Claudin-1, Nrf2, Keap-1, HO-1, Bax, Bcl-2, pro-Caspase 3, Cleaved-Caspase 3 were purchased from Affinity Company; β-actin was purchased from CellSignaling Technology Company; ultrasensitive ECL chemiluminescent liquid was purchased from Beijing Pulilai Gene Technology Co., Ltd.; BCA protein concentration determination kit was purchased from Shanghai Biyuntian Biotechnology Co., Ltd.; denatured protein precast gel (concentration 4-15%), Tris-glycine-SDS electrophoresis buffer (10×), TBS-T rinsing buffer (10×), SDS-PAGE protein loading buffer (5×), skim milk powder, Prestained Protein Marker (11-190 kDa) was purchased from Shanghai Yazyme Biotechnology Co., Ltd.; NC membrane was purchased from Wuhan Boster Bioengineering Co., Ltd.; Trizol was purchased from Thermo Fisher Scientific (China) Co., Ltd.; reverse transcription kit and qPCR kit were purchased from Hunan Aikerui Bioengineering Co., Ltd.; CAT, SOD, and kits were purchased from Beijing Solebow Technology Co., Ltd. (Beijing, China).

[0036] Example 1

[0037] (1) Experimental design

[0038] The experiment selected 48 PIC hybrid (28-day-old) weaned piglets with similar body weight (11.40±0.37) kg (provided by Sichuan Guotou Qiangshan Technology Group Co., Ltd.) and randomly divided them into 3 groups, 16 in each group. The control group (control) was fed with a basic diet, the low-dose group (YAML) was fed with 1% yam (Dioscorea oppositifolia, YAM) in the basic diet, and the high-dose group (YAMH) was fed with 2% yam in the basic diet. The yam used in the experiment was purchased from Anning Zhengdetang Medicinal Materials Store, Lanzhou City, Gansu Province.

[0039] (2) Basic diet and feeding management

[0040] Piglets were fed a basal diet prepared with corn-soybean meal. The nutrients and feed composition of the basal diet are shown in Table 1. Each group of pigs was housed individually in a pen with a stainless steel feeder and waterer. The room temperature was between 26 and 28 °C. In addition, the relative humidity level was maintained in the range of 65% to 75%. Feeding was done three times a day at 8:00, 14:00, and 20:00. During the 28-day trial, piglets had unlimited access to food and water. The animal research and related protocols used in this experiment were approved by the Animal Ethics Committee of the Lanzhou Institute of Animal Husbandry and Veterinary Medicine, Chinese Academy of Agricultural Sciences (License No.: SCXK Gansu 2023-021). The basal diet used in the experiment met the nutritional requirements recommended by the National Research Council of the United States (Table 1).

[0041] Table 1 Basic feed composition and nutritional level

[0042]

[0043]

[0044] 1 Premix provides: 9555IU vitamin A, 3000IU vitamin B1, vitamin C, vitamin B2, vitamin B3, vitamin B4, vitamin B6, vitamin B7, vitamin B8, vitamin B9, vitamin B12, vitamin B13, vitamin B14, vitamin B15, vitamin B16, vitamin B17, vitamin B18, vitamin B19, vitamin B12, vitamin B12, vitamin B12

[0045] Vitamin D3, 81IU Vitamin E, 3mg Vitamin K3, 3mg Vitamin B, 10.2mg Vitamin B2, 6mg Vitamin B6, 0.036mg Vitamin B12, 0.24

[0046] mg D-biotin, 30 mg D-pantothenic acid, 36 mg niacinamide, 300.06 mg choline chloride, 135 mg iron, 115 mg copper, 60 mg manganese, 1144.5 mg zinc, 118.2 mg

[0047] Methionine, 1638mg L-lysine.

[0048] 2 Calculated values ​​of each nutrient level.

[0049] (3) Sample collection

[0050] After 28 days of feeding, 15 mL of blood was collected from the anterior vena cava using a vacuum blood collection tube (without anticoagulant) and then allowed to rest for 10 min at 3500 r / min. -1Centrifuge for 10 min and take the supernatant for testing. After slaughter, 10-cm tissue segments were taken from the middle parts of the duodenum, jejunum, and ileum, respectively. Then, the intestinal lumen was thoroughly washed with ice-cold phosphate-buffered saline (PBS) solution. A 2-3-cm intestinal segment was taken and fixed in paraformaldehyde. The remaining tissue was placed in a 2-mL cryotube, immediately immersed in liquid nitrogen, and then stored at -80 °C for subsequent analysis. Samples for H&E staining and immunohistochemistry needed to be fixed in 4% paraformaldehyde.

[0051] Example 2

[0052] Effect of yam on antibody titer in the serum of weaned piglets

[0053] The antibody titer reflects the ability of a vaccine to induce specific immunity in an animal individual and is closely related to the health of the body to a certain extent. Classical swine fever (CSFV) and Swine pseudorabies (PRV) are highly prevalent diseases in nursery pigs.

[0054] (1) ELISA kit detection method:

[0055] According to the instructions of the ELISA kit (Jianglai Biotechnology Company), the contents of porcine pseudorabies virus antibody (PRV-Ab) and classical swine fever virus antibody (CSFV-Ab) in the serum were determined. All data were expressed as "Mean ± Standard Error of the Mean (Mean ± SEM)". The data were first tested for normal distribution and homogeneity of variance. If they conformed to normal distribution and had homogeneous variance, the One-Way ANOVA method was used for comparison between groups. If they did not conform to normal distribution, non-parametric tests were used. A trend of significant difference was considered when 0.05 < P < 0.10, and a significant difference was considered when P < 0.05. SPSS 27 was used for data statistical analysis, and GraphPad Prism 9 was used for graphing.

[0056] (2) Test results: As Figure 1 shown, compared with the Control group, during the entire test process, the antibody titers of CSFV-Ab and PRV-Ab showed an overall upward trend after adding yam. Among them, the antibody titers of CSFV-Ab and PRV-Ab in the YAMH group on the 14th day of the test were significantly higher than those in the control group (P < 0.05, Figure 1 A and B in). The results indicate that yam can increase the expression levels of CSFV-Ab and PRV-Ab in weaned piglets, suggesting that adding yam to the diet can improve the body's immune ability and disease resistance to CSFV and PRV.

[0057] Example 3

[0058] Effect of yam on intestinal mucosal immunity of weaned piglets

[0059] Defensins (pBDS) and interferons (IFNs) are closely related to intestinal mucosal immunity. Among them, pBDS is usually expressed in the intestinal mucosa and is the first line of defense for the host to resist invading pathogens. It is a key effector of innate immunity that can protect the host from pathogenic bacterial infections and regulate the composition of the intestinal microbiota.

[0060] (1) Detection method of gene expression

[0061] Add Trizol to intestinal tissues to extract total RNA. Use a ultra-micro spectrophotometer to detect the RNA concentration according to the instrument instructions and configure the reverse transcription system: 2 μL of 5×gDNA, 4 μL of 5×Evo M-mLVRT Reaction MIX, and a total of 14 μL of RNA + DEPC water to prepare a 20 μL reverse transcription system for reverse transcription. According to 5 μL of Green Pro Tap HS, 0.2 μL of each upstream and downstream primer, 4.1 μL of DEPC water, and 0.5 μL of cDNA to configure a 10 μL PCR system. Use a QuantStudio TM Design&Analysis real-time fluorescence quantitative PCR instrument for amplification, with β-actin as the internal reference. Pre-denature at 95 °C for 5 min, denature at 95 °C for 10 s, anneal at 60 °C for 20 s, extend at 72 °C for 20 s, for 40 cycles. Use the ΔCT method (2 -△△CT ) to calculate the change in CT value to judge the change in the expression of the target gene. The primer sequences are shown in Table 2.

[0062] (2) Data processing method

[0063] Each data is expressed as "mean ± standard deviation (Mean ± SEM)". The data are first tested for normal distribution and homogeneity of variance. If they conform to normal distribution and have homogeneous variance, the One-Way ANOVA method is used for comparison between groups. If they do not conform to normal distribution, non-parametric tests are used. 0.05 < P < 0.10 is considered to have a trend of significant difference, and P < 0.05 is considered a significant difference. SPSS 27 is used for data statistical analysis, and GraphPad Prism 9 is used for graphing.

[0064] Table 2 Primer sequences

[0065]

[0066]

[0067] (3) Experimental results

[0068] As Figure 2As shown in the figure, compared with the Control group, in the duodenum, the YAML group significantly increased the gene expression levels of pBD2, IFN-α and IFN-γ in the duodenum (P < 0.05), and the YAMH group significantly increased the gene expression levels of pBD1, pBD2, IFN-β and IFN-γ in the duodenum (P < 0.05). In the jejunum, the YAML group significantly increased the gene expression levels of pBD2, IFN-α and IFN-γ in the jejunum (P < 0.05), and the YAMH group significantly increased the gene expression levels of IFN-α, IFN-β and IFN-γ in the jejunum (P < 0.05). In the ileum, the YAML group significantly increased the gene expression levels of pBD2, IFN-γ and IFN-β in the duodenum (P < 0.05), and the YAMH group significantly increased the gene expression levels of pBD1, pBD2, IFN-α, IFN-β and IFN-γ in the ileum (P < 0.05). Therefore, adding yam to the diet of weaned piglets can enhance intestinal mucosal immunity by promoting the expression of intestinal defensins and interferon genes.

[0069] Example 4

[0070] Effects of Chinese Yam on Intestinal Barrier in Weaned Piglets

[0071] The intestinal mechanical barrier is the first line of defense to prevent harmful substances from invading the intestinal mucosa and destroying intestinal homeostasis. Tight junction proteins (TJs) are important intestinal mechanical barriers and have the function of preventing pathogen infection and invasion. Occludin, Claudin-1 and ZO-1 are the main cytoplasmic transmembrane and adapter proteins that form tight intercellular connections. Improving the expression of these key proteins can enhance the intestinal barrier function and reduce the permeability of the intestinal wall.

[0072] (1) Protein expression level detection:

[0073] Intestinal tissue was lysed with PMSF lysis buffer, and the supernatant was collected as a protein extract by centrifugation at 4°C and the concentration of the protein sample was measured according to the bicinchoninic acid assay (BCA). Equal amounts of protein samples were separated by 8-12% SDS-PAGE and then electrotransferred to polyvinylidene difluoride membranes (Millipore, MA, USA). The membranes were sealed with 5% skim milk at room temperature for 1.5 h and incubated with ZO-1 (1:1000), Occludin-1 (1:1000), and Claudin-1 (1:1000), respectively. After adding HRP-conjugated goat anti-mouse IgG secondary antibody (1:5000) or HRP-conjugated goat anti-rabbit IgG secondary antibody (1:5000), ultrasensitive ECL chemiluminescent solution (Apply.gen, Beijing, China) A solution and B solution 1 mL were mixed and dropped onto the NC membrane. The detection was performed using Biosciences Imager, and β-actin (1:1000) was used as a reference protein for comparison. The gray value of the bands was analyzed and calculated by Image J software.

[0074] (2) Detection of gene expression levels:

[0075] Trizol was added to the intestinal tissue to extract total RNA. The RNA concentration was detected by ultra-micro spectrophotometer according to the instrument instructions and the reverse transcription system was prepared: 2 μL of 5×gDNA, 4 μL of 5×Evo M-mLVRT Reaction MIX, and 14 μL of RNA+DEPC water were used to prepare 20 μL of reverse transcription system for reverse transcription. Green Pro Tap HS 5μL, upstream and downstream primers 0.2μL each, DEPC water 4.1μL, cDNA 0.5μL to prepare 10μL PCR system, use QuantStudio TM Design & Analysis real-time fluorescence quantitative PCR instrument was used for amplification, and β-actin was used as the internal reference. 95℃ pre-denaturation for 5 min, 95℃ denaturation for 10 s, 60℃ annealing for 20 s, 72℃ extension for 20 s, 40 cycles. The △CT method (2- △△ CT) was used to calculate the CT value change to determine the expression change of the target gene. The primer sequences are shown in Table 2.

[0076] (3) Data processing method:

[0077] Data are expressed as "Mean±SEM". Data were first tested for normal distribution and homogeneity of variance. One-Way ANOVA was used for comparison between groups when the data conformed to normal distribution and had homogeneous variance, and non-parametric tests were used when the data did not conform to normal distribution. A trend of significant difference was considered when 0.05 < P < 0.10, and a significant difference was considered when P < 0.05. SPSS 27 was used for data statistical analysis, and GraphPad Prism 9 was used for graphing.

[0078] (4) Experimental results

[0079] As Figure 3 shown. The results showed that the YAML and YAMH groups could significantly increase the protein expression levels of Claudin-1, Occudin-1, and ZO-1 in the duodenum (P < 0.05); in the jejunum, the YAML and YAMH groups could significantly increase the protein expression level of Occudin-1 in the jejunum (P < 0.05), and YAMH could significantly increase the protein expression levels of Claudin-1 and ZO-1 in the jejunum; in the ileum, the YAMH group could extremely significantly increase the protein expression levels of Occudin-1 and ZO-1 in the ileum (P < 0.01, Figure 3 A) in

[0080] By detecting at the gene level, it was found that compared with the Control group, the experimental group could significantly increase the gene expression levels of Claudin-1, Occudin-1, and ZO-1 in the duodenum (P < 0.05); similar results were obtained in the jejunum; in the ileum, the YAMH group significantly increased the gene expression levels of Claudin-1, Occudin-1, and ZO-1 (P < 0.05, Figure 3 B) in. The above results indicate that adding yam to the diet of weaned piglets can improve the intestinal barrier integrity and intestinal function of weaned piglets.

[0081] Example 5

[0082] Effect of yam on the Keap1 / Nrf2 / HO-1 antioxidant pathway in the intestine of weaned piglets

[0083] Weaning stress in piglets induces the production of a large amount of reactive oxygen species, which disrupts the intestinal oxidative balance and antioxidant system, thereby damaging the intestinal mucosa, causing intestinal damage and leading to intestinal barrier dysfunction, and a large amount of free radicals activate the classic Keap1 / Nrf2 / HO-1 antioxidant signaling pathway. The Keap1 / Nrf2 / HO-1 signaling pathway is a key pathway for regulating the antioxidant capacity of anti-oxidative stress, and is an important pathway for stimulating the expression of antioxidants and phase II enzymes. Usually, the transcription factor Nrf2 is combined with Keap-1 in the cytoplasm. When reactive oxygen species are produced in large quantities, Keap1 / Nrf2 dissociates, prompting Nrf2 to transfer from the cytoplasm to the nucleus to exert its effect, and then activates the expression of downstream antioxidant genes such as HO-1, Nqo1, CAT, and SOD.

[0084] (1) Protein expression level detection:

[0085] Intestinal tissue was lysed with PMSF lysis buffer, and the supernatant was collected as a protein extract by centrifugation at 4°C and the concentration of the protein sample was measured according to the bicinchoninic acid assay (BCA). Equal amounts of protein samples were separated by 8-12% SDS-PAGE and then electrotransferred to polyvinylidene difluoride membranes (Millipore, MA, USA). The membranes were sealed with 5% skim milk at room temperature for 1.5 h and incubated with Nrf2 (1:1000), Keap-1 (1:1000), HO-1 (1:1000), respectively, and HRP-conjugated goat anti-mouse IgG secondary antibody (1:5000) or HRP-conjugated goat anti-rabbit IgG secondary antibody (1:5000) was added. Then, 1 mL of A solution and B solution were mixed using ultrasensitive ECL chemiluminescent solution (Apply.gen, Beijing, China), and dropped onto the NC membrane. The detection was performed using Biosciences Imager, and β-actin (1:1000) was used as a reference protein for comparison. The gray value of the bands was calculated by Image J software.

[0086] (2) Detection of gene expression levels:

[0087] Trizol was added to the intestinal tissue to extract total RNA. The RNA concentration was detected by ultra-micro spectrophotometer according to the instrument instructions and the reverse transcription system was prepared: 2 μL of 5×gDNA, 4 μL of 5×Evo M-mLVRT Reaction MIX, and 14 μL of RNA+DEPC water were used to prepare 20 μL of reverse transcription system for reverse transcription. Green Pro Tap HS 5μL, upstream and downstream primers 0.2μL each, DEPC water 4.1μL, cDNA 0.5μL to prepare 10μL PCR system, use QuantStudio TMAmplification was performed using a Design&Analysis real-time fluorescence quantitative PCR instrument, with β-actin as the internal reference. Pre-denaturation at 95°C for 5 min, denaturation at 95°C for 10 s, annealing at 60°C for 20 s, extension at 72°C for 20 s, for 40 cycles. The ΔCT method (2 -△△CT ) was used to calculate the change in CT value to judge the change in the expression of the target gene, and the primer sequences are shown in Table 2.

[0088] (3) Data processing method:

[0089] All data were expressed as "mean ± standard deviation (Mean ± SEM)". The data were first subjected to normal distribution and homogeneity of variance tests. If they conformed to normal distribution and had homogeneous variance, the One-Way ANOVA method was used for comparison between groups. If they did not conform to normal distribution, non-parametric tests were used. A trend of significant difference was considered when 0.05 < P < 0.10, and a significant difference was considered when P < 0.05. SPSS 27 was used for data statistical analysis, and GraphPad Prism 9 was used for graphing.

[0090] (4) Experimental results:

[0091] As Figure 4 shown. As shown in A of Figure 4 . Compared with the Control group, the YAML group and the YAMH group could significantly increase the protein expression of Nrf2 and HO-1 in the duodenum and jejunum (P < 0.05). At the same time, the YAML group significantly decreased the protein expression level of Keap-1 in the jejunum (P < 0.05). In the ileum, the YAML group significantly increased the protein expression level of Nrf2 (P < 0.05), and the YAML group and the YAMH group significantly decreased the protein expression level of Keap-1 in the ileum and jejunum and significantly increased the protein ratio of HO-1 to reach the level (P < 0.05,).

[0092] As Figure 4 shown in B of

[0093] Example 6

[0094] Effect of Chinese Yam on Apoptosis of Intestinal Cells in Weaned Piglets

[0095] Oxidative stress is also closely related to the level of cell apoptosis. Superoxide anions and hydroxyl free radicals produced in large quantities in the body can induce cell apoptosis, and excessive apoptosis can cause damage to the intestinal mucosa. Bax, a pro-apoptotic gene in the Bcl-2 family of proteins, promotes cell apoptosis by combining with the anti-apoptotic gene Bcl-2 to form a dimer.

[0096] (1) Protein expression level detection:

[0097] Intestinal tissue was lysed with PMSF lysis buffer, and the supernatant was collected as a protein extract by centrifugation at 4°C and the concentration of the protein sample was measured according to the bicinchoninic acid assay (BCA). Equal amounts of protein samples were separated by 8-12% SDS-PAGE and then electrotransferred to a polyvinylidene difluoride membrane (Millipore, MA, USA). The membrane was sealed with 5% skim milk at room temperature for 1.5 h, and incubated with Bcl-2 (1:1000), Bax (1:1000), Cleaved-caspase-3 (1:1000) and Pro-caspase-3 (1:1000), respectively. After adding HRP-conjugated goat anti-mouse IgG secondary antibody (1:5000) or HRP-conjugated goat anti-rabbit IgG secondary antibody (1:5000), 1 mL of A solution and B solution were mixed using ultrasensitive ECL chemiluminescent solution (Apply.gen, Beijing, China), and dropped onto the NC membrane. Biosciences Imager was used for detection, and β-actin (1:1000) was used as a reference protein for comparison. The gray value of the band was analyzed and calculated by Image J software.

[0098] (2) Immunohistochemical staining:

[0099] HE sections were dewaxed into water, antigen repaired, and treated with 3% hydrogen peroxide. Circles were drawn and blocked with serum. Primary antibodies were incubated overnight at 4°C. Secondary antibodies were incubated at room temperature and color was developed. Immunohistochemical staining of cell nuclei was performed using hematoxylin staining, and the expression of apoptotic protein Bax was calculated using ImageJ software.

[0100] (3) Data processing method:

[0101] The Western blot images were processed using Image J. All data were expressed as "mean ± standard deviation (Mean ± SEM)". The data were first tested for normal distribution and homogeneity of variance. For data that conformed to normal distribution and had homogeneous variance, the One-Way ANOVA method was used for comparison between groups. For data that did not conform to normal distribution, non-parametric tests were used. A trend of significant difference was considered when 0.05 < P < 0.10, and a significant difference was considered when P < 0.05. SPSS 27 was used for data statistical analysis, and GraphPad Prism 9 was used for graphing.

[0102] (4) Test results:

[0103] As Figure 5 shown. In the duodenum, the YAML group significantly reduced the expression level of BAX protein, and the YAMH group significantly increased the expression level of Bcl-2 protein (P < 0.05). In the jejunum, both the YAML and YAMH groups significantly increased the expression level of Bcl-2 protein, and at the same time, the YAML group significantly reduced the expression level of Pro-caspase-3 protein (P < 0.05, 0.01). In the ileum, the YAML group significantly reduced the expression level of Bax protein (P < 0.05, 0.01), and the YAMH group significantly reduced the protein expression levels of Cleaved-caspase-3 and Pro-caspase-3 in weaned piglets (P < 0.05, Figure 5 as shown in A). The immunohistochemical results also showed that the addition of YAM could reduce the expression level of the pro-apoptotic protein Bax ( Figure 5 as shown in B). The results indicate that adding yam to the diet of weaned piglets can effectively inhibit the excessive apoptosis of intestinal mucosal epithelial cells caused by weaning.

[0104] As can be seen from the above examples, adding yam to the feed in the present invention can improve the immune capacity of weaned piglets and their disease resistance; enhance intestinal mucosal immunity by promoting the gene expression of intestinal defensins and interferons in weaned piglets; improve the integrity of the intestinal barrier and intestinal function of weaned piglets; enhance the antioxidant capacity of piglet intestines by activating the Keap1 / Nrf2 / HO-1 classical signaling pathway and increasing the expression of antioxidant enzymes; and can also effectively inhibit the excessive apoptosis of intestinal mucosal epithelial cells caused by weaning.

[0105] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. Application of yam in preparing feed for improving intestinal health of weaned piglets.

2. Application of yam in preparing feed for improving immunity of weaned piglets.

3. Application of yam in preparing feed for improving intestinal barrier integrity of weaned piglets.

4. Application of yam in preparing feed for improving the antioxidant capacity of weaned piglets.

5. The use of Chinese yam in preparing feed for improving the titer of serum antibodies in weaned piglets, characterized in that: The antibodies include CSFV-Ab and PRV-Ab.

6. The use of Chinese yam in preparing feed for improving the expression level of intestinal defensins and interferon genes in weaned piglets, characterized in that: The defensins include pBD1 and pBD2, and the interferons include IFN-α, IFN-β and IFN-γ.

7. Application of yam in preparing feed for improving the expression levels of Claudin-1, Occudin-1 and ZO-1 genes in the intestine of weaned piglets.

8. Use of Chinese yam in preparing feed for regulating Keap1 / Nrf2 / HO-1 antioxidant pathway in the intestine of weaned piglets, characterized in that: The expression levels of Nrf2 and HO-1 are increased, and the expression level of Keap1 is decreased; the Keap1 / Nrf2 / HO-1 antioxidant pathway increases the expression level of antioxidant enzymes and decreases the expression level of oxidative damage factors, the antioxidant enzymes include Nqo1, CAT, SOD2, and the oxidative damage factors include Nos2.

9. The use of Chinese yam in preparing feed for inhibiting intestinal cell apoptosis in weaned piglets, characterized in that: The apoptosis-related proteins include pro-apoptotic protein Bax and anti-apoptotic protein Bcl-2. The expression level of the pro-apoptotic protein Bax is reduced, and the expression level of the anti-apoptotic protein Bcl-2 is increased; the increased expression level of the anti-apoptotic protein Bcl-2 inhibits the expression of Cleaved-caspase-3, and the expression level of the Cleaved-caspase-3 protein is reduced.

10. The use according to any one of claims 1 to 9, characterized in that: The added amount of the yam is 1-2%.

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