A feed additive for improving intestinal morphology and barrier function of weaned piglets

Abstract

The application discloses a feed additive for improving intestinal morphology and barrier function of weaned piglets, and the feed additive is urolithin A. The application of urolithin A in the preparation of the feed additive for improving intestinal health of weaned piglets can effectively improve growth performance of the weaned piglets, improve final body weight and average daily gain of the piglets, and reduce feed conversion ratio. The application can improve intestinal morphology of the weaned piglets, increase intestinal villus height and villus crypt ratio, and reduce crypt depth. Meanwhile, the application can enhance intestinal barrier function and up-regulate mRNA expression levels of tight junction proteins ZO-1, Occludin and Claudin-1. The application provides a green new feed additive scheme for relieving weaning stress of piglets and maintaining intestinal health, and has a wide application prospect in pig breeding production.

Description

Technical Field

[0001] This invention belongs to the field of livestock feed technology, specifically relating to a feed additive that improves the intestinal morphology and barrier function of weaned piglets. Background Technology

[0002] my country is the world's largest pig-producing and pork-consuming country. The growth status of piglets during the weaning stage directly determines the production efficiency and overall economic benefits of pig farming. The intestine is the core site for nutrient digestion and absorption, and also the first line of defense for the body's immune system. It is the primary target organ for weaning stress. After weaning, piglets are prone to intestinal villus atrophy, crypt deepening, and a decreased villus height to crypt depth ratio, directly leading to a decline in intestinal digestive and absorptive capacity. Simultaneously, weaning stress disrupts the balance of intestinal epithelial cell renewal, downregulating the expression of tight junction proteins such as ZO-1, Occludin, and Claudin-1, resulting in increased intestinal permeability and impaired barrier function. This, in turn, triggers pathogen and toxin translocation, inducing local and even systemic inflammatory responses. Furthermore, weaning disrupts the intestinal microbiota structure, reducing the abundance of beneficial bacteria and increasing the proportion of opportunistic pathogens, further exacerbating intestinal inflammation and barrier damage. Against the backdrop of both antibiotic bans in feed and disease control, the development of safe, efficient, and green new feed additives has become an urgent need for the sustainable development of animal husbandry. Currently used intestinal regulators generally suffer from unstable effects and limited target sites, making it difficult to achieve the dual effects of intestinal morphology repair and barrier function enhancement simultaneously.

[0003] Urolithin A is a naturally occurring bioactive substance produced by the metabolism of ellagic acid and ellagitannins by intestinal microorganisms. It possesses good biocompatibility and chemical stability and has been widely used in food, medicine, and other fields. Existing research indicates that urolithin A plays an important role in regulating mitochondrial function, alleviating oxidative stress, inhibiting inflammatory responses, and maintaining tissue homeostasis. However, research and application of urolithin A in the regulation of intestinal health in pigs remain relatively scarce, particularly in improving intestinal morphology and enhancing intestinal barrier function, where systematic research and large-scale application plans are lacking. Summary of the Invention

[0004] The purpose of this invention is to provide a feed additive that improves the intestinal morphology and barrier function of weaned piglets. This invention, by adding urolithin A to the diet of weaned piglets, can effectively improve the integrity of the small intestinal morphology and structure, enhance intestinal barrier function, and thus promote the growth performance and intestinal health of weaned piglets.

[0005] The technical solution of the present invention is a feed additive that improves the intestinal morphology and barrier function of weaned piglets, wherein the feed additive is urolithin A.

[0006] The purity of the aforementioned feed additive, urolithin A, is not less than 96%.

[0007] Application of urolithin A in the preparation of feed additives to improve the intestinal health of weaned piglets.

[0008] In the aforementioned applications, the amount of urolithin A added to the feed is 50-200 mg / kg.

[0009] In the aforementioned applications, the amount of urolithin A added to the feed is 200 mg / kg.

[0010] In the aforementioned applications, the applications are used to improve the growth performance of weaned piglets, increase the final weight and average daily weight gain of weaned piglets, and reduce the feed conversion ratio.

[0011] In the aforementioned applications, the application is used to improve the intestinal morphology of weaned piglets, increase the intestinal villus height and villus-crypt ratio, and reduce crypt depth.

[0012] In the aforementioned applications, the application is used to enhance the intestinal barrier function of weaned piglets and upregulate the mRNA expression levels of tight junction proteins ZO-1, Occludin, and Claudin-1.

[0013] Compared with existing technologies, this invention uses urolithin A as a feed additive in pig farming, which can effectively improve the intestinal morphology and barrier function of weaned piglets and enhance growth performance. Adding an appropriate dose of urolithin A to the diet of weaned piglets can effectively increase the final body weight and average daily weight gain of piglets, and reduce the feed conversion ratio; it can also significantly increase jejunal villus height and villus-crypt ratio, reduce crypt depth, and improve the integrity of intestinal morphology and structure; at the same time, it can significantly upregulate the mRNA expression levels of tight junction proteins such as ZO-1, Occludin, and Claudin-1, enhancing intestinal barrier function. This invention provides a novel green feed additive solution for alleviating weaning stress and maintaining intestinal health in piglets, and has broad application prospects in pig farming. Attached Figure Description

[0014] Figure 1 The effect of adding urolithin A to feed on the intestinal morphology of weaned piglets. Detailed Implementation

[0015] The present invention will be further described below with reference to the accompanying drawings and embodiments, but this should not be construed as limiting the present invention.

[0016] Example: A feed additive for improving intestinal morphology and barrier function in weaned piglets, wherein the feed additive is urolithin A.

[0017] In this embodiment, 28 healthy Duroc × Landrace × Large White weaned piglets with an average weight of 8.56 kg were randomly divided into 4 treatment groups, with 7 replicates per treatment group and 1 piglet per replicate. The control group was fed a basal diet supplemented with 50 mg / kg, 100 mg / kg, and 200 mg / kg of urolithin A, respectively. Urolithin A (purity >96%) was purchased from Jinan Feiteng Pharmaceutical Technology Co., Ltd. The experiment was conducted at the Science and Education Training Base of the National Modern Animal Husbandry Demonstration Zone of Southwest University, with a duration of 21 days.

[0018] During the experiment, pigs had free access to feed and water, and were fed at fixed times at 08:00, 13:00, and 18:00 daily. Feed intake and health status of piglets were recorded. The experiment used a corn-soybean meal basal diet, formulated according to the "Chinese Swine Nutrition Requirements (2020)". The diet composition (%) was as follows: corn 66.58, soybean meal 7.5, fermented soybean meal 6.72, extruded soybean 6.9, whey powder 3.5, fish meal 3.5, soybean oil 1, lysine 0.4, methionine 0.03, threonine 0.12, tryptophan 0.05, dicalcium phosphate 1.2, limestone powder 1, salt 0.5, premix 1, total 100.

[0019] During the experiment, the initial weight, final weight, and feed intake of piglets were recorded, and the average daily feed intake, average daily weight gain, and feed conversion ratio were calculated. On the evening of day 21, all piglets were fasted for 12 hours. The following day, they were weighed, stunned by electric shock, and slaughtered by exsanguination. Intestinal tissue was quickly separated. The anterior jejunum was harvested; one portion was fixed in 4% paraformaldehyde for intestinal morphology analysis; the other portion was flash-frozen in liquid nitrogen and stored at -80°C for mRNA expression level detection. The 4% paraformaldehyde-fixed jejunum tissue was then washed, cleared, paraffin-embedded, trimmed, sectioned, and dewaxed before hematoxylin-eosin staining, dehydration, and mounting. The prepared sections were placed under an inverted optical microscope, and typical fields containing multiple complete villus morphologies were photographed. Image-Pro Plus 6.0 image analysis software was used to measure villus height and crypt depth, and the ratio of villus height to crypt depth (villus-crypt ratio) was calculated. Among them, villus height is defined as the distance from the villus tip to the midpoint of the line connecting the two ends of the crypt-villus junction; crypt depth is defined as the distance from the midpoint of the line connecting the two ends of the crypt-villus junction to the muscularis mucosae.

[0020] Total RNA was extracted from jejunal tissue using the Trizol method: Approximately 0.1 g of jejunal tissue sample stored at -80℃ was thoroughly ground in a sterile mortar and transferred to a 1.5 mL centrifuge tube. 1 mL of Trizol Reagent lysis buffer (Life Technologies) was added, and the mixture was vigorously vortexed to ensure complete lysis. Subsequent extraction procedures were performed according to the kit instructions, all under ice bath (4℃) conditions. 1 μL of total RNA was aspirated, and RNA purity was assessed using a P330 micro-spectrophotometer (Implen, Germany), ensuring an OD260 / OD280 ratio between 1.8 and 2.0. The purified RNA sample was then analyzed using PrimeScript. TM The RTReagentKit (TAKARA) reverse transcription kit was used to perform the reverse transcription reaction strictly according to the instructions to synthesize cDNA in a 20 μL reaction volume.

[0021] Based on the CDS sequences of porcine genes in NCBI GeneBank, qRT-PCR primers were designed and synthesized by Sangon Biotech (Shanghai) Co., Ltd., and diluted to 0.4 μM before use. Real-time quantitative PCR was performed using the SYBR Real-Time PCR kit (TAKARA) in 10 μL volumes, with three replicates for each target gene. The procedures were strictly followed according to the kit instructions.

[0022] This embodiment uses Microsoft Excel 2019 to organize experimental data, SPSS 19.0 statistical software for one-way ANOVA, and Duncan's method for multiple comparisons. Data are expressed as mean ± standard error (SEM). Linear and quadratic regression analyses were performed on different dose groups using orthogonal polynomial comparisons. P < 0.05 indicates statistical significance, and 0.05 ≤ P ≤ 0.10 indicates a significant trend.

[0023] The effects of urolithin A on the growth performance of weaned piglets are shown in Table 1. Compared with the control group, the addition of 50 mg / kg and 200 mg / kg urolithin A to the diet increased the final body weight of piglets by 5.47% and 4.87%, respectively, increased the average daily weight gain by 2.5% and 7.69%, respectively, and decreased the feed conversion ratio by 3.7% and 7.94%, respectively. The results indicate that adding 50-200 mg / kg urolithin A to the diet of weaned piglets can improve the final body weight and average daily weight gain, reduce the feed conversion ratio, and effectively improve the growth performance of weaned piglets.

[0024] Table 1. Effects of urolithin A supplementation on growth performance of weaned piglets. The effects of urolithin A on the intestinal morphology of weaned piglets are shown in Table 2. Figure 1 . Figure 1 The finger-like / leaf-like structures protruding into the intestinal lumen are intestinal villi, the core structure for intestinal digestion and nutrient absorption. The taller and more orderly the villi are, the larger the intestinal absorptive surface area and the stronger the function. The structures that are concave downwards at the base of the villi are intestinal crypts, the origin of intestinal epithelial cell renewal. The deeper the crypts, the stronger the compensatory proliferation after mucosal damage and the more obvious the intestinal stress damage. The ratio of villi height to crypt depth (villi-crypt ratio) is a core indicator for evaluating intestinal health; the higher the ratio, the healthier the intestinal morphology and the stronger the absorption function. Compared with the control group, dietary supplementation with 50, 100, and 200 mg / kg urolithin A significantly increased jejunal villus height (P < 0.05), with villus height increasing linearly with increasing dosage (P < 0.05). The crypt depth was significantly decreased in the 100 and 200 mg / kg urolithin A groups (P < 0.05), also decreasing linearly with increasing dosage (P < 0.05). The villus-crypt ratio was significantly increased in the 100 and 200 mg / kg urolithin A groups (P < 0.05), also increasing linearly with increasing dosage (P < 0.05). These results indicate that dietary supplementation with urolithin A can dose-dependently alleviate weaning stress-induced villus atrophy and crypt deepening in piglets, repair the integrity of the intestinal mucosa, and thus improve intestinal digestion and absorption capacity. This provides key morphological evidence supporting the core effects of this invention.

[0025] Table 2. Effects of urolithiasis A supplementation on intestinal morphology in weaned piglets. In Table 2, different lowercase letters in the same row heading indicate significant differences between treatment groups (P < 0.05); the same lowercase letter indicates no significant difference between treatment groups (P > 0.05).

[0026] The effects of urolithin A on the intestinal barrier function of weaned piglets are shown in Table 3. Compared with the control group, dietary supplementation with 200 mg / kg urolithin A significantly upregulated the mRNA expression levels of tight junction proteins ZO-1 and Occludin (P < 0.05); supplementation with 100 and 200 mg / kg urolithin A significantly upregulated the mRNA expression level of Claudin-1 (P < 0.05). The mRNA expression levels of ZO-1, Occludin, and Claudin-1 all increased linearly and significantly with increasing urolithin A dosage (P < 0.05). These results indicate that urolithin A can upregulate the expression of tight junction proteins and effectively enhance the intestinal barrier function of weaned piglets.

[0027] Table 3. Effects of urolithiasis A supplementation on intestinal barrier function in weaned piglets. In Table 3, different lowercase letters in the superscript of the same row indicate significant differences between treatment groups (P < 0.05); the same superscript letter indicates no significant difference between treatment groups (P > 0.05).

[0028] In summary, this invention, by adding urolithin A to the diet of weaned piglets, can effectively improve the integrity of the small intestinal morphology and structure, enhance the intestinal barrier function, and thus promote the growth performance and intestinal health of weaned piglets.

Claims

1. A feed additive for improving intestinal morphology and barrier function in weaned piglets, characterized in that, The feed additive is urolithiasis A.

2. The feed additive according to claim 1, characterized in that, The purity of the urolithin A is not less than 96%.

3. Application of urolithin A in the preparation of feed additives to improve the intestinal health of weaned piglets.

4. The application according to claim 3, characterized in that, The amount of urolithiasis A added to the feed is 50-200 mg / kg.

5. The application according to claim 4, characterized in that, The amount of urolithin A added to the feed is 200 mg / kg.

6. The application according to claim 3, characterized in that, The application is used to improve the growth performance of weaned piglets, increase the final weight and average daily weight gain of weaned piglets, and reduce the feed conversion ratio.

7. The application according to claim 3, characterized in that, The application is used to improve the intestinal morphology of weaned piglets, increase the height of intestinal villi and the villi-crypt ratio, and reduce crypt depth.

8. The application according to claim 3, characterized in that, The application is used to enhance the intestinal barrier function of weaned piglets and upregulate the mRNA expression levels of tight junction proteins ZO-1, Occludin, and Claudin-1.

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