Application of 1, 4-dihydroxy-2-naphthoic acid in improvement of animal growth performance and intestinal health

Abstract

The invention discloses application of 1, 4-dihydroxy-2-naphthoic acid in improvement of animal growth performance and intestinal health, and belongs to the technical field of feed additives. According to the invention, the 1, 4-dihydroxy-2-naphthoic acid is added into the basic feed, and tests prove that after the 1, 4-dihydroxy-2-naphthoic acid is added, the average daily gain and the average daily feed intake of broilers and piglets can be improved, and the feed conversion ratio is reduced; the piglet diarrhea can be improved, the development of the stomach and the small intestine of the piglet can be promoted, the morphological structure of the jejunum of the piglet can be improved, the development of the intestinal tract can be promoted, the activity of stem cells of the intestinal tract of the piglet can be regulated, and then the development of the gastrointestinal tract is promoted. Therefore, by adding the 1, 4-dihydroxy-2-naphthoic acid into the feed, the growth performance and the intestinal health of animals can be improved, the development of gastrointestinal tracts is promoted, the activity of the intestinal stem cells is regulated and controlled, and a new strategy is provided for improving the growth performance and the intestinal health of the animals.

Description

Technical Field

[0001] The invention relates to the technical field of feed additives, and in particular to application of 1,4-dihydroxy-2-naphthoic acid in improving animal growth performance and intestinal health. Background Art

[0002] The raising of piglets is an important link in the entire pig farming process. The survival rate and growth status of piglets are directly related to the economic benefits of farmers. Since the intestines and other organs of piglets are not yet fully developed, they are prone to problems such as indigestion, diarrhea, decreased feed intake, growth stunting, and even death, which brings significant economic losses to the pig farming industry and has become one of the important problems affecting the development of the pig farming industry.

[0003] The traditional solution to piglet diarrhea is to add antibiotics to feed. However, the use of antibiotics causes pathogen resistance and drug residues that seriously threaten human health. Currently, many countries have banned the addition of antibiotic growth promoters to feed. Existing alternatives to antibiotics mainly improve intestinal health by inhibiting the reproduction of pathogens such as E. coli. There is no particularly mature solution for promoting intestinal development to improve intestinal health and function in piglets.

[0004] 1,4-Dihydroxy-2-naphthoic acid is a synthetic precursor of vitamin K. It has functions such as regulating intestinal flora and improving colon inflammation. It is considered to be a potential drug for the treatment of human colon inflammation. However, there is no relevant report in the existing technology on whether it can improve the growth performance, intestinal health or promote gastrointestinal development of piglets and other animals. Summary of the invention

[0005] The purpose of the present invention is to provide an application of 1,4-dihydroxy-2-naphthoic acid in improving animal growth performance and intestinal health, so as to solve the problems existing in the above-mentioned prior art. The present invention adds 1,4-dihydroxy-2-naphthoic acid to feed to improve animal growth performance, intestinal health, promote gastrointestinal development and regulate intestinal stem cell activity, thereby providing a new strategy for improving animal growth performance and intestinal health.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The invention provides application of 1,4-dihydroxy-2-naphthoic acid in preparing a product for improving animal growth performance.

[0008] Optionally, the product is used to increase the average daily weight gain of animals and / or increase the average daily feed intake and / or reduce the feed-to-meat ratio.

[0009] Optionally, the animals include broiler chickens and piglets.

[0010] The invention also provides the use of 1,4-dihydroxy-2-naphthoic acid in preparing products for improving the intestinal health of animals.

[0011] Optionally, the product is used to improve diarrhea in piglets.

[0012] Optionally, the product is used to promote the development of the stomach and small intestine of piglets.

[0013] Optionally, the product is used to improve the morphological structure of piglet jejunum.

[0014] Optionally, the product is used to regulate the activity of intestinal stem cells in piglets.

[0015] Optionally, the product comprises a feed additive.

[0016] The present invention also provides a feed for improving animal growth performance and intestinal health, wherein the feed comprises 1,4-dihydroxy-2-naphthoic acid; the content of the 1,4-dihydroxy-2-naphthoic acid is 25 mg / kg-100 mg / kg.

[0017] The present invention discloses the following technical effects:

[0018] The present invention aims to solve the production problem that the intestinal development of piglets is not yet mature, which easily leads to intestinal health problems such as diarrhea, and provides a feed additive that promotes intestinal development and improves growth performance.

[0019] The present invention adds 1,4-dihydroxy-2-naphthoic acid to the basic feed. It has been verified through experiments that after adding 1,4-dihydroxy-2-naphthoic acid, the average daily weight gain and average daily feed intake of broilers and piglets can be increased, and the feed-to-meat ratio can be reduced; diarrhea in piglets can be improved, the development of the stomach and small intestine of piglets can be promoted, the morphological structure of the jejunum of piglets can be improved, the intestinal development can be promoted, and the activity of intestinal stem cells in piglets can be regulated, thereby promoting the development of the gastrointestinal tract. It can be seen that the addition of 1,4-dihydroxy-2-naphthoic acid to feed in the present invention can improve animal growth performance, intestinal health, promote gastrointestinal development and regulate intestinal stem cell activity, providing a new strategy for improving animal growth performance and intestinal health. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0021] Figure 1 This is a representative image of jejunal organoids treated with DHNA in the control group (Con);

[0022] Figure 2 This is a representative image of jejunal organoids treated with 2.5 μM DHNA;

[0023] Figure 3 This is a representative image of jejunal organoids treated with 5.0 μM DHNA;

[0024] Figure 4 This is a representative image of jejunal organoids treated with 10 μM DHNA;

[0025] Figure 5 Representative images of jejunal organoids treated with 20 μM DHNA. DETAILED DESCRIPTION

[0026] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0027] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0028] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0029] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0030] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0031] The 1,4-dihydroxy-2-naphthoic acid (DHNA) used in this example was purchased from Sigma-Aldrich.

[0032] Example 1 Poultry Experiment

[0033] 1. Animal grouping

[0034] 144 healthy 1-day-old white-feathered broilers were selected and randomly divided into 3 groups: the control group was fed with a basal diet (basic diet formula and nutritional components are shown in Table 1), and the other 2 groups were fed with a basal diet + 30 mg / kg DHNA and a basal diet + 80 mg / kg DHNA, respectively. Each group had 8 replicates, and each replicate had 6 chickens, for a total of 42 days. During the experiment, the feed intake of each cage of chickens was recorded every day, and each cage of chickens was weighed on the 1st, 21st and 42nd days of the experiment, and the average daily weight gain, average daily feed intake and feed-to-weight ratio were calculated. The result data were analyzed using SPSS software.

[0035] Table 1 Feed formula

[0036]

[0037]

[0038] 2. Experimental results and analysis

[0039] Adding 80 mg / kg DHNA to feed can increase the average daily gain (ADG) and average daily feed intake (ADFI) of white-feathered broilers aged 1-21 days, reduce the feed-to-meat ratio (FCR), and the average daily gain (ADG) of white-feathered broilers aged 22-42 days, indicating that adding DHNA to feed can improve the growth performance of white-feathered broilers.

[0040] Table 2 Effect of dietary DHNA on growth performance of white-feathered broilers

[0041] Comparison 30mg / kg 80mg / kg 1-21d ADG(g / d) <![CDATA[38.20 b ]]> <![CDATA[39.25 ab ]]> <![CDATA[42.87 a ]]> ADFI(g / d) <![CDATA[52.13 b ]]> <![CDATA[52.27 b ]]> <![CDATA[54.61 a ]]> FCR(d) <![CDATA[1.36 a ]]> <![CDATA[1.33 ab ]]> <![CDATA[1.27 b ]]> 22-42d ADG(g / d) <![CDATA[67.35 b ]]> <![CDATA[69.19 ab ]]> <![CDATA[71.36 a ]]> ADFI(g / d) 123.30 122.83 127.29 FCR(d) 1.83 1.77 1.78

[0042] Note: Different superscript letters in each row indicate statistically significant differences.

[0043] Example 2 Livestock Experiment

[0044] 1. Animal grouping

[0045] Thirty-two healthy 21-day-old piglets were selected and randomly divided into four groups according to their initial body weight: the control group was fed with a basal diet, the formula and nutritional components of the basal diet are shown in Table 3, and the other three groups were fed with a basal diet + 25 mg / kg, 50 mg / kg and 100 mg / kg of DHNA, 8 replicates per group, for a total of 21 days. During the experiment, feed intake, diarrhea score and other indicators were recorded every day, and the pigs were weighed on the 1st, 7th, 14th and 21st days of the experiment to calculate the average daily growth, average daily feed intake, feed-to-weight ratio, diarrhea index and diarrhea score, and the result data were analyzed using SPSS software.

[0046] Table 3 Basic diet raw material formula and nutritional components

[0047]

[0048]

[0049] 2. Test results and analysis

[0050] 2.1 Growth performance and diarrhea rate

[0051] As shown in Tables 5 and 6, adding DHNA to feed can increase the average daily weight gain (ADG) and average daily feed intake (ADFI) of piglets and reduce the feed-to-meat ratio (FCR), indicating that adding DHNA to feed can improve the growth performance of piglets. In addition, adding DHNA to feed can reduce the diarrhea rate and the number of piglets with diarrhea, and improve the intestinal health of piglets.

[0052] Among them, diarrhea rate = number of piglets with diarrhea during the experimental period / (number of experimental piglets × number of experimental days);

[0053] Diarrhea index = total diarrhea scores of experimental piglets during the experimental period / number of experimental days, where the diarrhea scoring standard is shown in Table 4.

[0054] Table 4 Scoring criteria for diarrhea

[0055]

[0056] Table 5 Effect of dietary DHNA on growth performance of piglets

[0057]

[0058] Note: Different superscript letters in each row indicate statistically significant differences.

[0059] Table 6 Effect of dietary DHNA on diarrhea rate and diarrhea index in piglets

[0060]

[0061] Note: Different superscript letters in each row indicate statistically significant differences.

[0062] 2.2 Organ index

[0063] The animals were slaughtered after 21 days of feeding, and the small intestine, large intestine and stomach were taken out, and their length and weight were measured to calculate the organ index (organ index = organ weight / body weight, organ length index = organ length / body weight).

[0064] As shown in Table 7, adding DHNA to the feed can increase the weight of the stomach and small intestine and the organ index of the stomach and small intestine of piglets, but has no obvious effect on the related indexes of the large intestine, indicating that adding DHNA to the feed can promote the development of the stomach and small intestine of piglets.

[0065] Table 7 Effects of dietary DHNA on gastrointestinal organ indexes in piglets

[0066]

[0067] Note: Different superscript letters in each row indicate statistically significant differences.

[0068] 2.3 Jejunal morphology and structure

[0069] The small intestine was taken to measure the height, width, surface area and crypt depth of the jejunal villi, and the villus-crypt ratio and villus surface area were calculated. The specific steps for jejunal morphological observation are as follows:

[0070] (1) Remove the jejunum from the 10% formalin solution, cut a regular sample in a fume hood, and place it in a pre-prepared embedding box. Then rinse the sample with running water overnight to remove the 10% formalin solution attached to the sample surface.

[0071] (2) The next day, the samples were dehydrated using gradient alcohol (85%, 95%, 100%) to remove excess water, and each gradient was soaked for 2 hours.

[0072] (3) The sample was then immersed in xylene until the tissue became transparent.

[0073] (4) Soak the transparent sample in paraffin wax, treat wax I and wax II for 1 hour each, and treat wax III for 40 minutes, and remove xylene. Then place the sample in the embedding box into the embedding mold, pour in the embedding wax, and wait for it to solidify.

[0074] (5) The jejunum embedded wax block was pre-frozen at -20°C, and then the sample was cut into 4 μm thick pieces using a microtome (RM2235, Leica, Germany). The sample cross section was kept intact during slicing, and then spread flat on the 42°C water surface of the microtome to make the sample cross section smoother for easier observation. Three discontinuous portions of each sample were placed on a glass slide. Subsequently, the sample was placed in a 37°C microtome for drying to remove moisture.

[0075] (6) For histological observation, the slide containing the tissue sample was first immersed in xylene for dewaxing. Then, it was rehydrated by soaking in graded alcohol (100%, 95%, 85%, 75%) (2, 1, 2, 2 min), and then soaked in double distilled water for 3 min to remove the alcohol. Then it was transferred to hematoxylin for staining for 7 min, and then rinsed with tap water for 5 min to remove excess stain. After that, it was decolorized by treating with 1% hydrochloric acid differentiation solution (4 mL concentrated hydrochloric acid and 396 mL 75% alcohol) for 30 s, then rinsed with tap water for 10 min, and then stained with eosin for 4-6 s. After staining, it was dehydrated again with graded alcohol (75%, 85%, 95%, 100%) (1, 1, 2, 2 min), and then transferred to xylene for transparent treatment. After the xylene was air-dried in the fume hood, it was sealed with neutral gum.

[0076] (7) The morphological structure of the jejunum was observed using an optical microscope (DM3000, Leica, Germany). Images were acquired using Leica Application Suite version 4.0 software. At least 30 complete villus-crypt structures were selected for measurement and analysis in each pig intestinal segment. Image J software was used to measure the villus height, crypt depth, and villus width of the jejunum, and the villus-crypt ratio and villus surface area were calculated.

[0077] As shown in Table 8, adding DHNA to feed can increase the height, width, villus crypt ratio and villus surface area of ​​the piglet jejunum, indicating that adding DHNA to feed can improve the morphological structure of the piglet jejunum and promote intestinal development.

[0078] Table 8 Effect of dietary DHNA on the intestinal morphology of piglets

[0079]

[0080] Note: The villus-crypt ratio is the ratio of villus height to crypt depth. The villus-crypt ratio is calculated for each individual and then the average value is calculated. Different superscript letters in each row indicate statistically significant differences.

[0081] Example 2 Organoid Test

[0082] (1) Select the proximal jejunum of the pig small intestine, remove the mesentery and cut along the longitudinal direction of the intestine. Then use pre-cooled PBS to flush the chyme and other intestinal contents to clean the intestine.

[0083] (2) Use a glass slide to gently scrape off the villi on the intestine and remove the fat. Then cut the intestine into tissue fragments of about 2 mm in size.

[0084] (3) The processed intestinal fragments were transferred to a 50 mL centrifuge tube and washed several times with pre-cooled PBS to remove contaminants and clearly expose the intestinal crypts. After the supernatant became clear, 2.5 mmol / L EDTA was added to the PBS and incubated on a shaker at 4°C for 30 min to separate the epithelium.

[0085] (4) After removing the dissociation reagent, PBS was added and pipetted 25 times to remove EDTA, thereby obtaining high-purity intestinal crypts.

[0086] (5) Filter using a 100 μm filter mesh, and then resuspend the crypts in a PBS solution containing 10% fetal bovine serum (FBS).

[0087] (6) The resuspension was centrifuged at 1000 rpm for 5 min, the supernatant was removed, and approximately 2 mL of complete culture medium was added.

[0088] (7) Centrifuge again at 600 rpm for 5 min, remove the supernatant, and add Matrigel to resuspend the crypts.

[0089] (8) Add 40 μL of the Matrigel-crypt mixture to the center of each well of a 24-well plate and place it at 37°C and 5% CO. 2 Then, a certain amount of culture medium containing various growth factors was added, including 100 ng / mL Wnt3a, 1 μg / mL R-spondin 1, 100 ng / mL Noggin, 2% B27, 1% N2, 10 mM nicotinamide, 50 ng / mL EGF, 1 mM n-acetyl-L-cysteine, 0.5 μM A83-01, 3 μM SB202190, and 2.5 μM CHIR99021.

[0090] Among them, Wnt3a, R-spondin 1, Noggin, B27 and N2 were purchased from Gibco; nicotinamide, EGF, n-acetyl-L-cysteine ​​and CHIR99021 were purchased from Sigma-Aldrich; A83-01 is a TGF-β inhibitor purchased from Tocris, Bristol, UK; SB202190 is a p38 inhibitor purchased from R&D Systems, Minneapolis, MN, USA.

[0091] (9) Treat organoids with DHNA solutions at concentrations of 0 μM, 2.5 μM, 5 μM, 10 μM, and 20 μM. Place the jejunal organoids in a 5% CO 2The cells were cultured in a 37°C incubator, wherein the 0 μM DHNA solution was the control group (CON).

[0092] (10) After the culture was completed, the organoids were observed and photographed using an optical microscope to analyze the bud length, number of buds, and area of ​​the organoids cultured at different concentrations, and the budding rate was calculated. The budding rate refers to the ratio of the number of budding organoids to the total number of organoids. The above indicators were measured using Image J software.

[0093] From Table 9 and Figure 1-Figure 5 It can be seen that within a certain concentration range, the addition of DHNA can increase the bud length of jejunal organoids as a whole, the addition of 2.5μM and 5μM DHNA can increase the budding rate of jejunal organoids, and the addition of DHNA will increase the area of ​​jejunal organoids as a whole, indicating that DHNA can regulate the activity of jejunal stem cells.

[0094] Table 9 Effects of DHNA on jejunal organoids

[0095]

[0096] Note: Different superscript letters in each row indicate statistically significant differences.

[0097] In summary, adding DHNA to feed can improve piglet growth performance, intestinal health, promote gastrointestinal development and regulate intestinal stem cell activity.

[0098] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. Application of 1,4-dihydroxy-2-naphthoic acid in the preparation of products for improving animal growth performance.

2. The use according to claim 1, characterized in that The product is used for increasing the average daily weight gain of animals and / or increasing the average daily feed intake and / or reducing the feed-to-meat ratio.

3. The use according to claim 1, characterized in that The animals include broiler chickens and piglets.

4. Application of 1,4-dihydroxy-2-naphthoic acid in the preparation of products for improving animal intestinal health.

5. The use according to claim 1, characterized in that The product is used for improving diarrhea in piglets.

6. The use according to claim 1, characterized in that The product is used to promote the development of the stomach and small intestine of piglets.

7. The use according to claim 1, characterized in that The product is used for improving the morphological structure of piglet jejunum.

8. The use according to claim 1, characterized in that The product is used for regulating the activity of intestinal stem cells in piglets.

9. The use according to any one of claims 1 to 8, characterized in that: The products include feed additives.

10. A feed for improving the growth performance and intestinal health of animal piglets, characterized in that: The feed contains 1,4-dihydroxy-2-naphthoic acid; the content of the 1,4-dihydroxy-2-naphthoic acid is 25 mg / kg-100 mg / kg.

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