Method for protecting intestinal immune function and inducing laying hen physiological remodeling through fasting

Abstract

The invention discloses a method for protecting intestinal immune function and inducing physiological remodeling of laying hens by fasting, belongs to the technical field of poultry farming, and provides a method for protecting intestinal immune function and inducing physiological remodeling of laying hens by fasting, which comprises the following steps: 1-12d before fasting of laying hens, the laying hens freely eat water and are drenched with Lactobacillus rhamnosus every day; fasting and water prohibiting are carried out on the first day to the third day of the fasting period; fasting and drinking water in the 4th-15th day of the fasting period, and drenching lactobacillus rhamnosus every day; and after the 15th day of the fasting period is finished, entering a recovery period, gradually increasing the feeding amount of the feed, and gradually recovering the illumination time. The lactobacillus rhamnosus is added in the fasting-induced physiological remodeling process, so that the fasting-induced intestinal injury of the laying hens can be improved.

Description

Technical Field

[0001] The invention belongs to the technical field of poultry breeding, and in particular relates to a method for fasting-induced physiological remodeling of laying hens to protect intestinal immune function. Background Art

[0002] After poultry go through an egg-laying cycle, their bodies consume a lot of nutrients, their physical fitness declines, and their egg production and quality begin to decline, eventually leading to normal physiological molting. Therefore, most commercial laying hens or breeding hens will be eliminated when their egg production rate drops to a level that is not enough to bring benefits to the breeding company in the first egg-laying cycle.

[0003] In nature, birds need to go through a natural molting process before winter arrives, shedding old feathers and growing new feathers to cope with the cold winter climate. Molting usually occurs in autumn, during which the food intake of birds is greatly reduced, ovarian function declines, and body weight decreases by about 30% to 40%. The natural molting period lasts about 3 to 4 months and is associated with a decrease in the egg production rate of hens and a decrease in eggshell quality. For commercial laying hens, their economic value will drop significantly after natural molting. In order to maintain an uninterrupted supply of eggs, many farmers choose to sell and replace hens before they molt naturally. Fasting-induced physiological remodeling is a comprehensive regulatory technology for laying / breeding hens in the late egg-laying period. It stimulates and activates the natural physiological resting law of poultry through dietary and environmental regulation, so that they stop laying eggs / sperm in a concentrated manner, and smoothly enter a new round of reproductive cycle after resting. It is an important technology for the extended rearing of laying / breeding hens and has broad application prospects in laying / breeding hens production.

[0004] Based on the results of previous studies, fasting can cause oxidative damage in the intestine (jejunum and ileum) mediated by increased MDA content, increased intestinal permeability caused by decreased intestinal ZO-1, Occludin-1, and Claudin-1 mRNA expression, increased mRNA expression of inflammatory cytokines IL-1β, IL-8, and NF-kB, intestinal villus atrophy, epithelial cell shedding, inflammatory cell infiltration in the lamina propria, and other pathological phenomena. Fasting induction can reduce the species richness and uniformity of the intestinal flora of laying hens and change the structure of the intestinal flora. During the fasting period, the abundance of probiotics such as Bacteroides, Faecalibacterium, Lactobacillus, and Paralactobacillus in the cecum decreased, while the abundance of harmful bacteria such as Desulfovibrio, Clostridium, Escherichia Castellani and Chalmers increased, which is closely related to impaired intestinal barrier function, decreased immunity, and the occurrence of intestinal inflammatory diseases. With the resumption of feeding, intestinal damage and intestinal flora composition returned to the level before fasting. Therefore, according to the changes in intestinal flora structure and metabolites during fasting-induced physiological remodeling, the targeted addition of probiotics and other microecological preparations to regulate the intestinal microecology and the production of short-chain fatty acids is an important solution to intestinal health. Summary of the invention

[0005] In order to solve the above technical problems, the present invention proposes a method for fasting-induced physiological remodeling of laying hens to protect intestinal immune function, and Lactobacillus rhamnosus is added during the fasting-induced physiological remodeling process. This method has an improving effect on fasting-induced intestinal damage in laying hens.

[0006] To achieve the above object, the present invention provides a method for fasting-induced physiological remodeling of laying hens for protecting intestinal immune function, comprising the following steps:

[0007] (1) Laying hens were allowed to eat and drink freely from 1 to 12 days before fasting, and were gavaged with Lactobacillus rhamnosus daily.

[0008] (2) During the first 1 to 3 days of the fasting period, no food or water is allowed;

[0009] (3) During the 4th to 15th day of the fasting period, fasting was allowed, water was available freely, and Lactobacillus rhamnosus was administered orally daily;

[0010] (4) After the 15th day of the fasting period, the recovery period begins, during which the amount of feed fed is gradually increased and the lighting time is gradually restored.

[0011] Preferably, the laying hens in step (1) are laying hens with a daily egg production rate of less than 40%, and the laying hens are bantam chickens.

[0012] Preferably, the daily dosage of Lactobacillus rhamnosus administered orally in step (1) is 1 mL / each, and the effective viable count of Lactobacillus rhamnosus in step (1) is greater than 1×10 9 CFU / mL.

[0013] Preferably, the light exposure is maintained at 16 h / d during the 1st to 12th day before the fasting in step (1).

[0014] Preferably, during the first to third days of the fasting period in step (2), light is maintained at 8 h / d.

[0015] Preferably, during the 4th to 15th day of the fasting period in step (3), light is maintained at 8 h / d.

[0016] Preferably, the daily dosage of Lactobacillus rhamnosus administered orally in step (3) is 1 mL / each, and the effective viable count of Lactobacillus rhamnosus is greater than 1×10 9 CFU / mL.

[0017] Preferably, the recovery period in step (4) is 30 days.

[0018] Preferably, the gradually increasing feed amount in step (4) is as follows: 25-30 g / head per day on the 1st to 2nd day of the recovery period, 50-60 g / head per day on the 3rd to 4th day of the recovery period, 75-90 g / head per day on the 5th day of the recovery period, 100-120 g / head per day on the 6th to 17th day of the recovery period, and free diet is resumed on the 17th to 30th day of the recovery period.

[0019] Preferably, the step of gradually restoring the illumination time in step (4) is as follows: during the recovery period, the illumination time is increased by 0.5 h per day until the illumination time reaches 16 h / d, and then the illumination time is kept unchanged.

[0020] Compared with the prior art, the present invention has the following advantages and technical effects:

[0021] The present invention adds Lactobacillus rhamnosus (LGG) during the process of fasting-induced physiological remodeling. Lactobacillus rhamnosus is widely used as a probiotic for humans or livestock due to its resistance to gastric acid and bile and its strong affinity for intestinal mucosal cells. Lactobacillus rhamnosus can not only produce antibacterial substances to stabilize the intestinal microecological environment, but also directly participate in the repair of host intestinal mucosal barrier function damage and intestinal mucosal immune regulation to maintain intestinal health. The intestinal damage before and after fasting was detected, and the results showed that the addition of Lactobacillus rhamnosus can improve the antioxidant capacity and immune performance of laying hens with physiological remodeling, improve intestinal barrier function, thereby reducing intestinal inflammation, and further improve the recovery ability of the intestine of laying hens with fasting-induced physiological remodeling. In summary, the addition of Lactobacillus rhamnosus during fasting-induced physiological remodeling has an improving effect on fasting-induced intestinal damage in laying hens. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] 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.

[0023] Figure 1 To calculate the weight loss rate during fasting-induced physiological remodeling;

[0024] Figure 2 To calculate the weight recovery rate during fasting-induced physiological remodeling;

[0025] Figure 3 To calculate the egg-laying rate during fasting-induced physiological remodeling;

[0026] Figure 4 The intestinal tissue (H&E) staining images during the F0 period of fasting-induced physiological remodeling, where A is the jejunum of the NC group, B is the ileum of the NC group, C is the jejunum of the TB group, and D is the ileum of the TB group. The scale bar is 200 μm.

[0027] Figure 5 The statistical graphs of the villus height, crypt depth and villus-crypt ratio of the jejunum and ileum during the F0 period of fasting-induced physiological remodeling, wherein A is the quantitative result of the jejunal villus height, B is the quantitative result of the ileal villus height, C is the quantitative result of the jejunal crypt depth, D is the quantitative result of the ileal crypt depth, E is the quantitative result of the jejunal villus-crypt ratio, and F is the quantitative result of the ileal villus-crypt ratio;

[0028] Figure 6Figure 2 is the intestinal tissue (H&E) staining image at F15 during the fasting-induced physiological remodeling process, where A is the jejunum of the NC group, B is the ileum of the NC group, C is the jejunum of the TB group, D is the ileum of the TB group, E is the jejunum of the TF group, F is the ileum of the TF group, G is the jejunum of the TBF group, and H is the ileum of the TBF group. The scale bar is 200 μm.

[0029] Figure 7 The statistical graphs of villus height, crypt depth and villus-crypt ratio of jejunum and ileum at F15 period during the fasting-induced physiological remodeling process, wherein A is the quantitative result of jejunal villus height, B is the quantitative result of ileal villus height, C is the quantitative result of jejunal crypt depth, D is the quantitative result of ileal crypt depth, E is the quantitative result of jejunal villus-crypt ratio, and F is the quantitative result of ileal villus-crypt ratio;

[0030] Figure 8 The intestinal tissue (H&E) staining images at R5 during the fasting-induced physiological remodeling process, where A is the jejunum of the NC group, B is the ileum of the NC group, C is the jejunum of the TB group, D is the ileum of the TB group, E is the jejunum of the TF group, F is the ileum of the TF group, G is the jejunum of the TBF group, and H is the ileum of the TBF group. The scale bar is 200 μm.

[0031] Fig. 9 The statistical graphs of villus height, crypt depth and villus-crypt ratio of jejunum and ileum at R5 period during fasting-induced physiological remodeling, among which A is the quantitative result of jejunal villus height, B is the quantitative result of ileal villus height, C is the quantitative result of jejunal crypt depth, D is the quantitative result of ileal crypt depth, E is the quantitative result of jejunal villus-crypt ratio, and F is the quantitative result of ileal villus-crypt ratio;

[0032] Fig.10 Figure 2 is the intestinal tissue (H&E) staining image at R30 during the fasting-induced physiological remodeling process, where A is the jejunum of the NC group, B is the ileum of the NC group, C is the jejunum of the TB group, D is the ileum of the TB group, E is the jejunum of the TF group, F is the ileum of the TF group, G is the jejunum of the TBF group, and H is the ileum of the TBF group. The scale bar is 200 μm.

[0033] Fig.11 The statistical graphs of villus height, crypt depth and villus-crypt ratio of jejunum and ileum at R30 period during the fasting-induced physiological remodeling process, wherein A is the quantitative result of jejunal villus height, B is the quantitative result of ileal villus height, C is the quantitative result of jejunal crypt depth, D is the quantitative result of ileal crypt depth, E is the quantitative result of jejunal villus-crypt ratio, and F is the quantitative result of ileal villus-crypt ratio;

[0034] Fig.12The effect of adding Lactobacillus rhamnosus on inflammatory factors during fasting-induced physiological remodeling, among which A is jejunal IL-1β, B is ileal IL-1β, C is jejunal IL-6, D is ileal IL-6, E is jejunal IL-8, F is ileal IL-8, G is jejunal TGF-β3, and H is ileal TGF-β3;

[0035] Fig.13 The effect of adding Lactobacillus rhamnosus on tight junction proteins during fasting-induced physiological remodeling, where A is jejunal ZO-1, B is ileal ZO-1, C is jejunal Claudin-1, D is ileal Claudin-1, E is jejunal Occludin-1, and F is ileal Occludin-1;

[0036] Fig.14 This is the effect of adding Lactobacillus rhamnosus on proliferation- and apoptosis-related genes during fasting-induced physiological remodeling, among which A is jejunal CDK1, B is ileal CDK1, C is jejunal LGR5, D is ileal LGR5, E is jejunal MYC, F is ileal MYC, G is jejunal BCL2, and H is ileal BCL2. DETAILED DESCRIPTION

[0037] 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.

[0038] It should be understood that the terms described in the present invention are only for describing special embodiments 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. Each smaller range between the intermediate value in any stated value or stated range and 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.

[0039] 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.

[0040] 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 the skilled artisan. The present invention description and examples are exemplary only.

[0041] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0042] Example 1

[0043] 1 Materials and methods

[0044] 1.1 Animals and experimental design

[0045] The experimental animals were provided by the Poultry Germplasm Resource Factory of Henan Agricultural University. The study was conducted from July to October 2023. During the feeding experiment, the indoor temperature was maintained at 18-25°C and the relative humidity was maintained at 60%-70%. A total of 288 420-day-old bantam chickens were selected and randomly divided into 4 groups: NC group, TB group, TF group and TBF group. NC group (no oral administration of Rhamnosus bacillus, 1 mL of normal saline was administered to each chicken every day from the 1st to the 12th day before fasting; and on the 4th to 15th day of the fasting period, 1 mL of normal saline was administered to each chicken every day during the fasting period), TB group (the number of effective live bacteria was greater than 1×10 9 CFU / mL of Lactobacillus rhamnosus was administered to each chicken by gavage every day from 1 to 12 days before fasting, and 1 mL of saline was administered to each chicken by gavage every day from 4 to 15 days during the fasting period and during the fasting period), and TF group (1 mL of saline was administered to each chicken by gavage every day from 1 to 12 days before fasting; and 1 mL of effective viable bacteria count was greater than 1×10 9 CFU / mL of Lactobacillus rhamnosus 1 mL per chicken), TBF group (the number of effective live bacteria was greater than 1×10 CFU / mL per day on the 1st to 12th day before fasting) 9 CFU / mL of Lactobacillus rhamnosus 1mL per chicken; and during the 4th to 15th day of the fasting period, the effective live bacteria count per day should be greater than 1×10 9 CFU / mL of Lactobacillus rhamnosus 1 mL per chicken), and the Lactobacillus rhamnosus (ATCC53103) used in this experiment was provided by BNCC (BNCC, Xinyang, China).

[0046] The experimental adaptation period was 3 days, and the experimental period (1st to 12th day before fasting, 15th day of fasting period and 30th day of recovery period) was 57 days. The fasting-induced physiological remodeling program included: 1st to 12th day before fasting (free feeding, 16h / d of light), 15 days of fasting period (1st to 3rd day, fasting and water deprivation, 4th to 15th day, fasting and water deprivation, 8h / d of light per day), 30 days of recovery period (feeding amount according to: 25g / head on the first day of recovery period, 30g / head on the second day of recovery period, 50g / head on the third day of recovery period, 60g / head on the fourth day of recovery period, 90g / head on the fifth day of recovery period, 100g / head on the sixth day of recovery period, 102g / head on the seventh day of recovery period, 8h / head on the eighth day of recovery period). Feed 104g / head, on the 9th day of the recovery period, feed 106g / head every day, on the 10th day of the recovery period, feed 108g / head every day, on the 11th day of the recovery period, feed 110g / head every day, on the 12th day of the recovery period, feed 112g / head every day, on the 13th day of the recovery period, feed 114g / head every day, on the 14th day of the recovery period, feed 116g / head every day, on the 15th day of the recovery period, feed 118g / head every day, on the 16th day of the recovery period, feed 120g / head every day, on the 17th day of the recovery period, feed 120g / head every day, and resume free diet from the 17th to the 30th day of the recovery period; light is increased by 0.5h in the morning and afternoon alternately until 16h / d). Monitor the changes in body weight and mortality rate. If the weight loss rate reaches 25% to 30% or the mortality rate is greater than 2%, fasting should be stopped in advance. Chickens were killed by bleeding from the jugular vein one day before fasting (F0), on the 15th day of fasting (F15), on the 5th day of recovery (R5), and on the 30th day of recovery (R30). Serum was collected, and fixed samples of the jejunum and ileum were collected and stored in centrifuge tubes containing 4% paraformaldehyde for subsequent sectioning. Tissue samples of the jejunum and ileum were collected and placed in 1.5 mL sterile enzyme-free centrifuge tubes, quickly frozen in liquid nitrogen, and finally stored in a -80°C refrigerator for later use. The composition and nutritional level of the basic diet are shown in Table 1.

[0047] Table 1 Composition and nutritional level of basic diet feed

[0048]

[0049] Note: a premix provides the following elements per kilogram of daily feed: Vitamin A, 518320IU; Vitamin D, 100026IU; 25-hydroxyvitamin D3, 3137μg; Vitamin E, 4550IU; Vitamin K3, 272.8mg; Vitamin B1, 136.4mg; Vitamin B2, 750.2mg; Vitamin B6, 272.8mg; Vitamin B12, 1.4mg; D-biotin, 16.4mg; D-pantothenic acid, 818.4mg; Folic acid, 136.4mg; Nicotinamide, 2728mg; Choline chloride, 25000mg; Iron, 3000mg; Copper, 454.0mg; Manganese, 5454mg; Zinc, 4770mg; Iodine, 91.0mg; Selenium, 13.6mg; Methionine, 55000mg; Moisture content is not higher than 10%.

[0050] bExcept for metabolizable energy, all other indicators are measured values.

[0051] 1.2 Determination of egg laying performance and egg quality

[0052] Eggs were collected manually at 14:00 every day, and the number of eggs laid by each group was counted for the calculation of egg production rate. At F0 and R30, 30 eggs were collected from each group for egg quality testing, and the differences in egg quality among the groups were compared. The egg shape index (major axis / minor axis) was measured with a vernier caliper. The eggshell thickness was measured using an eggshell thickness meter (robotation ETG-1601A; Nanjing, China). The eggshell strength was measured using an eggshell strength meter (robotation EFG-0503; Tokyo, Japan). The eggshell weight was measured using an analytical balance. The remaining indices were measured using a multifunctional egg analyzer (robotation EMT-5200; Tokyo, Japan).

[0053] 1.3 Intestinal morphology analysis

[0054] After fixation with 4% paraformaldehyde for 24 h, the intestinal tissue was embedded in paraffin and sliced ​​at 5 μm using a microtome. All sections were stained with hematoxylin and eosin (H&E) for morphological observation of the jejunum and ileum. At least 6 to 10 villi were selected for each tissue section, and the average value was taken as the sample measurement value. Motic DSAssistant Lite software was used to calculate the villus height and crypt depth.

[0055] 1.4 Immunity performance index detection

[0056] The levels of immunoglobulin A (IgA), immunoglobulin G (IgG), and immunoglobulin M (IgM) in serum samples were detected using ELISA kits purchased from Jiangsu ELISA Industrial Co., Ltd. (Jiangsu, China). All experimental steps were performed according to the instructions of the kits.

[0057] 1.5 Oxidation and anti-oxidation index detection

[0058] The levels of T-AOC, SOD, MDA, CAT, and GSH-PX in serum and jejunal tissue were detected using biochemical kits purchased from Jiangsu Enzyme Immunity Industry Co., Ltd. (Jiangsu, China). All experimental steps were performed according to the instructions of the kits.

[0059] 1.6 Fluorescence quantitative PCR detection

[0060] Tissue RNA was extracted from jejunal and ileal tissues using TRIzol reagent (Vazyme, Nanjing, China). RNA concentration was measured using a NanoDrop ND2000 spectrophotometer (NanoDrop Products, Wilmington, NC, USA) according to the manufacturer's instructions. RNA samples that met the requirements were reverse transcribed. RNA was reverse transcribed into cDNA using HiScript III RT SuperMix for qPCR (Vazyme, Nanjing, China). The resulting product was diluted with 60 μL of sterile enzyme-free water and stored at -20°C for later use. Real-time quantitative PCR (qRT-PCR) was used with 2 -ΔΔCT The mRNA expression levels of inflammatory factors, tight junction proteins, and proliferation and apoptosis-related genes in the jejunum and ileum were evaluated by the method. qRT-PCR was performed on the LightCycler96 Real-Time PCR System (Roche, Basel, Switzerland) using the ChamQ Universal SYBR qPCR Master Mix Kit (Vazyme, Nanjing, China). The reaction system of 10 μL included: 1 μL cDNA, 5 μL 2×ChamQ Universal SYBR qPCR Master Mix, 0.5 μL of upstream and downstream primers, and 3 μL sterile enzyme-free water. The cycling program of qRT-PCR included: 95℃5min, then 95℃10s followed by 60℃30s for 40 cycles, then 95℃10s, 60℃60s, and finally 95℃ for 15s. The primer sequences are shown in Table 2.

[0061] Table 2 Primer sequences

[0062]

[0063]

[0064] Note: In the table, F represents the upstream primer sequence, and R represents the downstream primer sequence.

[0065] 1.7 Data Statistical Analysis

[0066] SPSS26.0 and GraphPad Prism 8 were used to perform one-way or two-way ANOVA and LSD test for all experimental data. When the data did not conform to normal distribution, KruskalWallis test was used. All data are expressed as Mean±SEM. "*" indicates P<0.05, "**" indicates P<0.01, and "***" indicates P<0.001. Data with different superscript letters (a, b, c) are statistically significant (P<0.05).

[0067] 2 Results and analysis

[0068] 2.1 Adding Lactobacillus rhamnosus can improve the production performance of laying hens with physiological remodeling

[0069] like Figure 1 As shown in Figure 2, during the fasting-induced physiological remodeling process, the body weight of each group decreased fastest at F3, and the weight loss rate reached the maximum at F15. Figure 2 As shown in the figure, the body weight recovered rapidly after the resumption of feeding, and the recovery rate reached the maximum at R24, reaching about 96.5% of the initial body weight. The subsequent body weight remained basically stable, and the difference in body weight changes among the four groups was not large. Figure 3 As shown, the initial egg production rate of each group was about 30%. Egg production stopped from F7 during the fasting period and resumed from R19. The TBF group started laying eggs earlier than the control group. At R32, the egg production rates of the TB and TBF groups were about 25% higher than those of the control group.

[0070] Table 3 Effect of adding Lactobacillus rhamnosus on mortality rate during fasting-induced physiological remodeling

[0071]

[0072] Table 4 Effect of adding Lactobacillus rhamnosus on egg quality during diet-induced physiological remodeling

[0073]

[0074]

[0075] The effect of adding Lactobacillus rhamnosus on the mortality rate during the fasting-induced physiological remodeling process is shown in Table 3. The mortality rates of each group during the adaptation period were basically the same. The mortality rate of the control group during the fasting period was much higher than that of the three treatment groups. There was no death in the recovery treatment group. The effect of adding Lactobacillus rhamnosus on egg quality during the fasting-induced physiological remodeling process is shown in Table 4. During the F0 period, the egg shape index of the control group and the TF group was significantly higher than that of the TB group and the TBF group (P<0.05). During the R30 period, the eggshell weight of the TF group was significantly higher than that of the TB group, the albumen height of the TBF group was significantly higher than that of the other three groups, and the yolk color of the TF group was significantly lighter (P<0.05). In summary, these data show that the addition of Lactobacillus rhamnosus during the fasting physiological remodeling process can improve egg quality to a certain extent.

[0076] 2.2 Adding Lactobacillus rhamnosus can improve the physiological remodeling of the intestinal morphology of laying hens

[0077] Effects of the addition of Lactobacillus rhamnosus on intestinal morphology during fasting-induced physiological remodeling, e.g. Figure 4 Middle A, Figure 4 Middle B, Figure 4 Middle C and Figure 4 As shown in D, the jejunal and ileal tissues at F0 are intact and neatly arranged, and the lamina propria is compactly arranged. Figure 5 Middle A, Figure 5 Middle B, Figure 5 Middle C, Figure 5 Middle D, Figure 5 ZhongE and Figure 5 As shown in F, compared with the NC group, the height of villus and the villus-to-crypt ratio of the jejunum in the TB group were significantly different (P<0.05), but there was no significant difference between the ileum.

[0078] like Figure 6 Middle A, Figure 6 Middle B, Figure 6 Middle C, Figure 6 Middle D, Figure 6 Middle E, Figure 6 Medium F, Figure 6 Medium G and Figure 6 As shown in Figure H, the inflammatory cell infiltration in the lamina propria was more serious at F15, and there was severe shedding of the villus tips. Figure 7 Middle A, Figure 7 Middle B, Figure 7 Middle C, Figure 7 Middle D, Figure 7 ZhongE and Figure 7 As shown in F, compared with the NC group, the situation in the TBF group was improved, and the villus height of the jejunum was significantly increased (P<0.01), and the villus-crypt ratio was significantly increased (P<0.05), but there was no significant difference between the groups in the ileum.

[0079] like Figure 8 Middle A, Figure 8 Middle B, Figure 8 Middle C, Figure 8 Middle D, Figure 8 Middle E, Figure 8 Medium F, Figure 8 Medium G and Figure 8 As shown in H, the tissue structure is relatively intact at R5. Although inflammatory cell infiltration occurs, the degree is milder than that at F15. Fig. 9 Middle A, Fig. 9 Middle B, Fig. 9 Middle C, Fig. 9 Middle D, Fig. 9 ZhongE and Fig. 9 As shown in F, compared with the NC group, the TBF group had the best recovery, and there was a very significant difference in the villus height of the jejunum (P<0.01), and a significant difference in the crypt depth (P<0.05), but there was no significant difference between the groups in the ileum.

[0080] like Fig.10 Middle A, Fig.10 Middle B, Fig.10 Middle C, Fig.10 Middle D, Fig.10 Middle E, Fig.10 Medium F, Fig.10 Medium G and Fig.10 As shown in H, the jejunal and ileal tissues at R30 were intact and neatly arranged, and the lamina propria was compactly arranged. Fig.11 Middle A, Fig.11 Middle B, Fig.11 Middle C, Fig.11 Middle D, Fig.11 ZhongE and Fig.11 As shown in F, compared with the NC group, only the TF group had a significantly higher chorionic villus ratio (P<0.05), while the other two groups showed no significant changes.

[0081] These data suggest that the decline in production performance caused by fasting may be due to the reduction in villus height and increase in crypt depth, and that the addition of Lactobacillus rhamnosus during fasting-induced physiological remodeling could improve the intestinal absorptive capacity by increasing the height of jejunal villi and reducing the depth of crypts, thereby ameliorating the intestinal damage caused by fasting.

[0082] 2.3 Addition of Lactobacillus rhamnosus can improve physiological remodeling of the intestinal chemical barrier of laying hens

[0083] Effects of adding Lactobacillus rhamnosus during fasting-induced physiological remodeling on inflammatory factors, tight junction proteins, and proliferation- and apoptosis-related genes, e.g. Figures 12-14 As shown. Fig.12 Middle A, Fig.12 Middle B, Fig.12 Middle C, Fig.12Middle D, Fig.12 Middle E, Fig.12 Medium F, Fig.12 Medium G and Fig.12 As shown in Figure H, compared with NC, the pro-inflammatory factors IL-1β, IL-6, and IL-8 in the jejunum of the TBF group decreased significantly before and after fasting, and the anti-inflammatory factor TGF-β3 increased significantly in the late recovery period. The pro-inflammatory factors IL-1β and IL-6 in the ileum decreased significantly (P<0.05), and the contents of the pro-inflammatory factor IL-8 and the anti-inflammatory factor TGF-β3 in the ileum did not change significantly in the two groups. Fig.13 Middle A, Fig.13 Middle B, Fig.13 Middle C, Fig.13 Middle D, Fig.13 ZhongE and Fig.13 As shown in Figure F, tight junction proteins Claudin-1 and Occludin-1 were significantly increased in the jejunum and ileum (P<0.05), while ZO-1 was significantly different only in the jejunum. Fig.14 Middle A, Fig.14 Middle B, Fig.14 Middle C, Fig.14 Middle D, Fig.14 Middle E, Fig.14 Medium F, Fig.14 Medium G and Fig.14 As shown in Figure H, the proliferation-promoting genes CDK1, LGR5, and MYC increased significantly in the jejunum (P<0.05), and the apoptosis-inhibiting gene BCL2 also increased significantly (P<0.05), but only CDK1 and MYC increased significantly in the ileum (P<0.05). The results show that the addition of Lactobacillus rhamnosus during fasting-induced physiological remodeling can reduce intestinal inflammatory response, improve intestinal barrier function, and promote intestinal epithelial cell proliferation and differentiation.

[0084] 2.4 Addition of Lactobacillus rhamnosus can improve the antioxidant level of physiologically remodeled laying hens

[0085] The effects of adding Lactobacillus rhamnosus on the oxidation and antioxidant levels of the jejunum during fasting-induced physiological remodeling are shown in Table 5. During the fasting-induced physiological remodeling, the MDA content in the jejunum of the NC group showed a trend of first increasing and then decreasing, while the levels of T-AOC, SOD, and GSH-PX showed the opposite trend. However, CAT activity increased to a certain extent during the fasting-induced physiological remodeling. In order to verify the accuracy of the data in the jejunum, the results were verified with serum, and the oxidation and antioxidant levels in the serum were similar to those in the jejunum, as shown in Table 6. It is worth noting that compared with the NC group, the MDA content in the TBF group was significantly reduced, and the activities of T-AOC and CAT were significantly increased (P<0.05). These data prove that Lactobacillus rhamnosus, on the one hand, reduces the oxidative damage caused by fasting by reducing the MDA content during fasting-induced physiological remodeling, and on the other hand, resists the stress caused by fasting by increasing the activity of antioxidant enzymes.

[0086] Table 5 Effects of adding Lactobacillus rhamnosus on jejunal oxidation and antioxidant levels during fasting-induced physiological remodeling

[0087]

[0088]

[0089] Table 6 Effects of adding Lactobacillus rhamnosus on serum oxidation and antioxidant levels during fasting-induced physiological remodeling

[0090]

[0091] 2.5 Adding Lactobacillus rhamnosus can improve the immune level of laying hens with physiological remodeling

[0092] The effect of adding Lactobacillus rhamnosus on serum immune level during fasting-induced physiological remodeling is shown in Table 7. The expression of IgA, IgG and IgM in the NC group and TBF group showed a trend of first decreasing and then increasing during fasting-induced physiological remodeling, and decreased significantly after fasting. Compared with the NC group, the content of IgA, IgG and IgM in the TBF group during fasting-induced physiological remodeling was significantly increased (P<0.05). In summary, the addition of Lactobacillus rhamnosus during fasting-induced physiological remodeling can improve the immune level of laying hens.

[0093] Table 7 Effect of adding Lactobacillus rhamnosus on serum immune levels during fasting-induced physiological remodeling

[0094]

[0095] Therefore, in the process of fasting-induced physiological remodeling, adding Lactobacillus rhamnosus (LGG) can help repair intestinal damage in laying hens, improve antioxidant and immune capabilities, enhance intestinal barrier function, and reduce inflammation. In particular, adding Lactobacillus rhamnosus in the early and middle stages of fasting is most beneficial to the intestinal health of laying hens.

[0096] The results showed that 1) compared with the NC group, the egg production rate of the TB group and the TBF group was significantly increased (P<0.05), while the egg production rate of the TF group did not change significantly (P>0.05); the villus-crypt ratio of the jejunum in the TB group increased significantly at F0 (P<0.05), the villus-crypt ratio of the jejunum in the TBF group increased significantly at F0 and F15 (P<0.05), and the villus-crypt ratio of the jejunum and ileum in the TF group did not change significantly (P>0.05). In the TBF group, the villus height of the jejunum and ileum increased significantly at R5 (P<0.05), but the crypt depth of the jejunum also increased significantly (P<0.05). Based on the above results, the differences between the NC group and the TBF group were compared. 2) Compared with the NC group, the levels of IgA, IgG, and IgM in the serum of the TBF group increased significantly (P<0.05); the level of MDA decreased significantly (P<0.05); the activities of T-AOC and CAT increased significantly (P<0.05), and the results in the jejunum were similar to those in the serum. The pro-inflammatory factors IL-1β and IL-6 in the jejunum and ileum decreased significantly (P<0.05), and the anti-inflammatory factor TGF-β3 in the jejunum increased significantly (P<0.05); the tight junction proteins Claudin-1 and Occludin-1, the proliferation-promoting gene CDK1, and the apoptosis-inhibiting gene MYC in the jejunum and ileum also increased significantly (P<0.05). In summary, the addition of Lactobacillus rhamnosus during the fasting-induced physiological remodeling process has an ameliorative effect on fasting-induced intestinal damage in laying hens, and the TBF group is superior to the TB and TF groups.

[0097] 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. A method for fasting-induced physiological remodeling of laying hens for protecting intestinal immune function, characterized in that: The following steps are involved: (1) Laying hens were allowed to eat and drink freely from 1 to 12 days before fasting, and were gavaged with Lactobacillus rhamnosus every day. (2) During the first 1 to 3 days of the fasting period, no food or water is allowed; (3) During the 4th to 15th day of the fasting period, fasting was allowed, water was available freely, and Lactobacillus rhamnosus was administered orally daily; (4) After the 15th day of the fasting period, the recovery period begins, during which the amount of feed fed is gradually increased and the lighting time is gradually restored.

2. The method according to claim 1, characterized in that: The laying hens described in step (1) are laying hens with a daily egg production rate of less than 40%, and the laying hens are bantam chickens.

3. The method according to claim 1, characterized in that: The dosage of Lactobacillus rhamnosus administered orally in step (1) is 1 mL / each, and the effective viable count of Lactobacillus rhamnosus in step (1) is greater than 1×10 9 CFU / mL.

4. The method according to claim 1, characterized in that: During the first to 12 days before the fasting described in step (1), the light exposure was maintained at 16 h / d.

5. The method according to claim 1, characterized in that: During the first to third days of the fasting period in step (2), the light intensity was maintained at 8 h / d.

6. The method according to claim 1, characterized in that: During the 4th to 15th day of the fasting period in step (3), the light intensity was maintained at 8 h / d.

7. The method according to claim 1, characterized in that: The dosage of Lactobacillus rhamnosus administered daily in step (3) is 1 mL / each, and the effective viable count of Lactobacillus rhamnosus is greater than 1×10 9 CFU / mL.

8. The method according to claim 1, characterized in that: The recovery period in step (4) is 30 days.

9. The method according to claim 1, characterized in that: The step (4) of gradually increasing the amount of feed is as follows: 25-30 g / head per day on the 1st to 2nd day of the recovery period, 50-60 g / head per day on the 3rd to 4th day of the recovery period, 75-90 g / head per day on the 5th day of the recovery period, 100-120 g / head per day on the 6th to 17th day of the recovery period, and free diet is resumed on the 17th to 30th day of the recovery period.

10. The method according to claim 1, characterized in that: The step of gradually restoring the illumination time in step (4) is as follows: during the recovery period, the illumination time is increased by 0.5 h per day until the illumination time reaches 16 h / d, and then the illumination time is kept unchanged.

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