Application of all-transretinoic acid in preparation of feed for improving pulmonary nerve development of pig fetus

Abstract

The invention discloses application of all-transretinoic acid in preparation of feed for improving lung nerve development of a pig fetus, and belongs to the technical field of livestock breeding and feed. 4-32 mg / kg of all-trans retinoic acid (ATRA) is added into the complete formula feed, development of astrocytes and microglia in lung tissues of the piglets is regulated and controlled through maternal nutrition, lung health of the newborn piglets and excitability of lung nerves are improved, the newborn piglets can inhale more oxygen to meet various needs of organisms, and the feed has the advantages of being capable of improving the immunity of the newborn piglets and improving the survival rate of the newborn piglets. Therefore, the disease resistance and growth intensity of the organism are enhanced.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of livestock breeding and feed, and particularly relates to application of all-trans retinoic acid in preparation of feed for improving pig fetal lung nerve development. BACKGROUND

[0002] The lung is a key organ for gas exchange and immune defense in animals, and lung dysfunction can lead to respiratory diseases or increased susceptibility to diseases, thereby impairing its respiratory and immune functions, ultimately causing animals to have abnormal breathing, decreased disease resistance, growth retardation, or even death. Lung function is closely related to the development of the pulmonary nervous system, because lung tissue contains a large amount of peripheral nerve tissue, which plays an important role in regulating lung health and function. Studies have shown that the pulmonary nervous system is composed of neurons and glial cells, and the neurons in the lung tissue mainly function in the regulation of the lung, detecting information such as chemical concentration and mechanical pressure in the lung through sensory nerve endings, and transmitting these information to the central nervous system. At the same time, motor nerve fibers can control respiration and gland secretion by regulating the contraction and relaxation of airway and alveolar smooth muscle. Glial cells are an indispensable component of the peripheral nervous system, and they bear important regulatory functions for the health and function of neurons. These glial cells are mainly responsible for supporting, nourishing and protecting neurons; they help neurons absorb nutrients and remove metabolic waste, and participate in the metabolism and circulation of neurotransmitters, thereby regulating the efficiency of nerve signal transmission. In addition, glial cells also have specific immune defense functions, and when lung tissue is damaged or infected, they can participate in the regulation of inflammatory response to protect neural tissue from further damage.

[0003] At present, the rate of scale of pig farms in China has reached more than 75%, and the breeds raised are all foreign breeds (Changbai, Dabai and Duroc); in scale pig farms, the morbidity and mortality caused by lung diseases (cough, panting, inflammation and congestion and swelling, etc.) of pigs are very high, and the lung diseases of pigs are closely related to abnormal lung nerve development during the fetal period of pigs. In view of this, how to improve the lung nerve development of pig fetuses has become a key problem to be solved. SUMMARY

[0004] The application provides application of all-trans retinoic acid in preparation of feed for improving pig fetal lung nerve development, and aims to solve the problems in the background art.

[0005] In order to achieve the above technical purposes, the technical scheme mainly adopted by the application is as follows: In a first aspect, the application discloses application of all-trans retinoic acid in preparation of feed for improving pig fetal lung nerve development; the improvement of pig fetal lung nerve development is achieved by increasing the number of astrocytes and the number of astrocyte-neuron co-localization in lung nerve tissue, increasing the number of microglia cells and nerve fiber cells, or regulating the nerve active ligand-receptor interaction pathway and the gamma-aminobutyric acid synapse pathway.

[0006] As a preferred embodiment, the feed for improving pig fetal lung nerve development is a complete formula feed added with 4-32 mg / kg all-trans retinoic acid.

[0007] As a preferred embodiment, the complete formula feed comprises: corn 50%-60%, soybean meal 15%-17%, beet pulp 10%-15%, wheat bran 3%-5%, and premix 4%-5%.

[0008] As a preferred embodiment, the pig fetus is a Duroc, Landrace, and Large White three-way cross pig fetus.

[0009] As a preferred embodiment, the feed for improving pig fetal lung nerve development is fed from the 12th day to the 95th day after the sow is bred, and an ordinary daily ration is fed at other times until the sow gives birth.

[0010] In a second aspect, the application discloses application of all-trans retinoic acid in preparation of feed for increasing the number of astrocytes and the number of astrocyte-neuron co-localization in lung nerve tissue of newborn piglets.

[0011] In a third aspect, the application discloses application of all-trans retinoic acid in preparation of feed for increasing the number of microglia cells and nerve fiber cells.

[0012] In a fourth aspect, the application discloses application of all-trans retinoic acid in preparation of feed for regulating the nerve active ligand-receptor interaction pathway and the gamma-aminobutyric acid synapse pathway.

[0013] Traditional research believes that there are only three kinds of glial cells in lung nerve tissue, namely Schwann cells, satellite cells, and enteric glial cells, while astrocytes and microglia cells only exist in the central nervous system. However, the latest research reports point out that in addition to Schwann cells and satellite glial cells, there are also astrocytes and microglia cells in peripheral nerve tissue. The specific marker gene of Schwann cells is S100, the preferred marker gene of satellite glial cells is FABP7, astrocytes can be specifically labeled by the GFAP gene, and the characteristic marker of microglia cells is TMEM119.

[0014] Currently, there are no studies on the regulation of astrocyte and microglia development in lung tissue through maternal nutrition. A significant increase in the number of astrocytes and a marked decrease in the expression of inhibitory neurotransmitter receptor genes play a crucial role in enhancing the health and function of neurons in lung tissue. This helps improve lung health and pulmonary nerve excitability in newborn piglets, allowing them to inhale more oxygen to meet their various needs, thereby enhancing their disease resistance and growth rate.

[0015] Compared with the prior art, the beneficial effects of the present invention are: This invention uses Duroc-Landrace-Large White three-way crossbred pig fetuses as a model to study the effects of feeding different doses of all-trans retinoic acid (ATRE) to pregnant sows at specific stages (fetal lung development stage) on astrocytes, microglia, neurons, and nerve fibers in the lung tissue of newborn piglets. This provides technical support for reducing lung-related diseases caused by incomplete development of the pulmonary nervous system in pig fetuses, and also provides a reference for research on the prevention and control of lung diseases in human newborns. Attached Figure Description

[0016] Figure 1 This study compares the expression of astrocytes and neurons in the lung tissue of newborn piglets treated with different doses of all-trans retinoic acid.

[0017] Figure 2 This study compares the expression of microglia and nerve fibers in the lung tissue of newborn piglets treated with different doses of all-trans retinoic acid. Detailed Implementation

[0018] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below in conjunction with embodiments. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0019] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0020] Example 1 Experimental Design: 1. Artificial insemination was performed on 15 crossbred (Landrace and Large White) sows of similar body condition, age, and parity using the same Duroc boar semen. After mating, these sows were randomly assigned to 5 all-trans retinoic acid (ATRA) treatment groups: ATRA0 group (complete feed without ATRA, composed of: corn 59%, soybean meal 16.5%, beet meal 15%, wheat bran 5%, premix 4.5%, the same below), ATRA4 group (complete feed with ATRA added at 4 mg / kg), ATRA8 group (complete feed with ATRA added at 8 mg / kg), ATRA16 group (complete feed with ATRA added at 16 mg / kg), and ATRA32 group (complete feed with ATRA added at 32 mg / kg). From day 12 to day 95 post-mating, sows in the ATRA0, ATRA4, ATRA8, ATRA16, and ATRA32 treatment groups were fed complete feed with the corresponding ATRA-added amount. For the remaining time, sows in all treatment groups were fed complete feed without ATRA until farrowing. Each treatment group had three replicates, with one pregnant sow per replicate. Specific experimental treatments are shown in Table 1.

[0021] Table 1 Experimental Design

[0022] 2. Before the newborn piglets nurse, select two piglets (one male and one female) from each litter whose weight is closest to the average. Euthanize them using sodium pentobarbital (100 mg / kg body weight). Open the thoracic cavity, remove the lungs, and collect lung tissue samples from the same location in the right lung lobe of each piglet. One portion of the samples is fixed in 4% paraformaldehyde solution, while the other portion is flash-frozen in liquid nitrogen and preserved. Used for testing other indicators in an 80℃ ultra-low temperature freezer.

[0023] Lung tissue samples were fixed in 4% paraformaldehyde solution for 24 hours at 3-4℃, then embedded in paraffin. The paraffin-embedded samples were then cut into 5 µm thick sections and fixed onto glass slides. After dewaxing, dehydration, antigen retrieval, and blocking with 10% donkey serum, the slides were incubated overnight at 4℃ with the specified primary antibodies (GFAP: Oasis biofarm, 1:200; β3-tubulin: Abcam,ab18207, 1:2000; TMEM119: Oasis biofarm, OB-PGP072-02, 1:300; PGP 9.5:Starter, S0B2306, 1:300). After washing with PBS (phosphate-buffered saline, pH 7.4), slides were incubated with designated secondary antibodies (Alexa Fluor® 594 donkey anti-rabbit IgG (H+L): ThermoFisher, A21207, 1:600; Alexa Fluor® 488 donkey anti-rabbit IgG (H+L): ThermoFisher, A21205, 1:400; donkey anti-guinea pig IgG, AF594: Oasisbiofarm, D-GP594-50, 1:400) at 37°C for 45 minutes. After washing again with PBS, the slides were stained with DAPI (4', 6-diamidinyl-2-phenylindole, Solarbio, C0060). Stained samples were scanned using a Pannoramic MIDI scanner (3Dhistech Ltd., Budapest, Hungary), and target images were captured using Case Viewer software (3D-histech Ltd., Budapest, Hungary).

[0024] 4. Total RNA was extracted from lung tissue samples using the TRNzol universal kit (Tiangen Biotech, Beijing, China). RNA sequencing libraries were constructed using the Fast RNA-seq Library Preparation Kit V2 (ABclonal, Wuhan, China). RNA sequencing was performed using an Illumina NovaSeq 6000 sequencing platform (San Diego, California, USA). Raw reads were filtered and pruned using fastp software (version 0.19.7), and then clean reads were aligned to the reference genome using HISAT2. The expression levels of each gene were determined by calculating the number of fragments per kilobase transcript per million aligned fragments (FPKM) and using Cufflinks. Differentially expressed genes (DEGs) were screened using DESeq 2 software with a p-value < 0.05 and a |log2 fold change| > 2 as the threshold. The function of DEGs was investigated using KEGG pathway enrichment analysis.

[0025] Experimental results: 1. Effects of pregnant sows' intake of complete feed supplemented with different doses of all-trans retinoic acid on the number of astrocytes (GFAP) and neurons (TUBB3) in the lung nerve tissue of newborn piglets. Figure 1 The results showed that, compared with the control group (ATRA0), large white and white crossbred sows fed a complete feed containing 4 mg of all-trans retinoic acid per kilogram (ATRA4 treatment group) from day 12 to day 95 post-mating had a significantly increased number of astrocytes in the lung nerve tissue of newborn piglets. Figure 1 F) and the number of astrocyte-neuron colocalizations ( Figure 1 H), but regarding the number of neurons ( Figure 1 G) No significant effect.

[0026] 2. Effects of pregnant sows' intake of complete feed supplemented with different doses of all-trans retinoic acid on the number of microglia (TMEM119) and nerve fibers (PGP9.5) in the lung nerve tissue of newborn piglets. Figure 2 Data showed that, compared with the control group (ATRA0), large white and white crossbred sows fed a complete feed containing 32 mg of all-trans retinoic acid per kilogram (ATRA32 treatment group) from day 12 to day 95 post-mating had a significantly increased number of microglia in the lung nerve tissue of newborn piglets. Figure 2 F) had no significant effect on the number of nerve fibers. Figure 2 G); The ATRA8 treatment group significantly increased the number of microglia co-localized with nerve fibers in the lung tissue of newborn piglets (G); Figure 2 H).

[0027] 3. Effects of diets supplemented with different doses of all-trans retinoic acid on neural pathways and gene expression in the lung tissue of newborn piglets in pregnant sows. Table 2. Effects of pregnant sows' consumption of complete feed containing different doses of all-trans retinoic acid on neural pathways and gene expression in the lung tissue of newborn piglets.

[0028] Table 2 shows that, compared with the control group (ATRA0), the consumption of complete feed containing all-trans retinoic acid by Landrace and Large White sows from day 12 to day 95 post-mating significantly affected gene expression in the neuroactive ligand-receptor interaction pathway and the GABAergic synapse pathway.

[0029] The experimental results showed that supplementing pregnant sows with a complete feed containing 4 mg / kg all-trans retinoic acid from day 12 to day 95 after mating could significantly increase the number of astrocytes and the number of astrocyte-neuron colocalizations in the pulmonary nerve tissue; at the same time, it could also increase the number of microglia and nerve fiber cells to varying degrees, and improve the immunity and excitability of the pulmonary nerves.

[0030] The embodiments described above are merely preferred embodiments of the present invention, and while the descriptions are specific and detailed, they are not intended to limit the present invention. It should be noted that various changes and modifications can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the concept and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. Application of all-trans retinoic acid in the preparation of feed that improves the development of the pulmonary nerves in pig fetuses.

2. The application according to claim 1, characterized in that, The feed that improves the development of the pulmonary nerves in pig fetuses is a complete compound feed supplemented with 4-32 mg / kg all-trans retinoic acid.

3. The application according to claim 2, characterized in that, The complete compound feed includes: 50%-60% corn, 15%-17% soybean meal, 10%-15% beet meal, 3%-5% wheat bran, and 4%-5% premix.

4. The application according to claim 1, characterized in that, The pig fetuses mentioned are Duroc, Landrace, and Large White three-way crossbred pig fetuses.

5. The application according to claim 1, characterized in that, From day 12 to day 95 after mating, sows were fed the feed that improves the development of the pulmonary nerves of the pig fetus; otherwise, they were fed a regular diet until farrowing.

6. Application of all-trans retinoic acid in the preparation of feed that increases the number of astrocytes and the number of astrocyte-neuron colocalizations in the lung nerve tissue of newborn piglets.

7. Application of all-trans retinoic acid in the preparation of feeds that increase the number of microglia and nerve fiber cells.

8. Application of all-trans retinoic acid in the preparation of feeds that regulate neuroactive ligand-receptor interaction pathways and γ-aminobutyric acid synaptic pathways.

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