Application of protein zinc with medium chelating strength in improvement of heat stress resistance of chicken

Abstract

The invention discloses an application of protein zinc with medium chelating strength in improvement of heat stress resistance of chicken, and provides a new function of protein zinc with medium chelating strength, and zinc is added under a heat stress condition to relieve small intestine mucosa injury of heat stress broiler chicken, the medium-chelating-strength protein zinc is added into the feed, so that the relieving effect on the heat-stress small intestinal mucosal injury of the broiler chicken is obvious; wherein the 60mg Zn / kg feed obtained by supplementing the protein zinc with medium chelating strength has a more obvious effect of relieving the heat stress small intestine mucosa injury of the broiler chicken; specifically, the permeability of each section of the small intestine of the broiler chicken is reduced, and the barrier function is enhanced. Therefore, the supplement of the protein zinc with medium chelating strength in the feed is most beneficial to the improvement of the small intestine mucosa injury of the heat stress broiler chicken. The protein zinc with medium chelating strength can be effectively used for preparing a feed additive for improving the heat stress resistance of chicken and preparing a feed additive for improving the small intestine mucosa injury of heat stress chicken.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of livestock and poultry breeding, and relates to the use of a novel organic zinc product. Specifically, in the case that chickens are in a heat stress state, a medium chelation strength protein zinc is selected as a novel zinc source additive for additional zinc supplementation to improve the heat stress resistance of the chickens. BACKGROUND

[0002] Broiler chickens grow fast, have high feed conversion efficiency, and are the world's largest meat product due to their high protein content and low saturated fatty acid content. Broiler production is highly intensive, and because of their fast growth, high metabolism, and high body temperature, they have no sweat glands, so they are very sensitive to heat stress (Sahin et al., Role of dietary zinc in heat-stressed poultry: a review, Poultry Science, 2009; Impact of heat stress on poultry health and performances, and potential mitigation strategies, Animals, 2020). The intestinal tract of broiler chickens and other livestock is the central organ of the stress response, and is particularly sensitive to heat stress (Nanto-Hara et al., Heat stress directly affects intestinal integrity in broiler chickens, The Journal of Poultry Science, 2020). Previous studies have found that heat stress can reduce the ratio of small intestinal villus height to crypt depth and villus surface area in broiler chickens, increase the crypt depth of the duodenum, jejunum, and ileum, and lead to increased intestinal permeability and serum endotoxin content (Santos et al., Quantitative histo-morphometric analysis of heat-stress-related damage in the small intestines of broiler chickens, Avian Pathology, 2015; Alhenaky et al., The effect of heat stress on intestinal integrity and salmonella invasion in broiler birds. Journal of Thermal Biology, 2017). Previous studies have shown that heat stress inhibits the expression of the tight junction proteins occludin and zonula occludens-1 (ZO-1) in the ileal mucosa of broiler chickens (Tabler et al., Intestinal barrier integrity in heat-stressed modern broilers and their ancestor wild jungle fowl, Frontiers in Veterinary Science, 2020).Nanto-Hara et al. (2020) found that heat stress reduced the number of Proliferating Cell Nuclear Antigen (PCNA) positive cells in the duodenum of broilers (Nanto-Hara et al., Effects of dietary brown rice on the growth performance, systemic oxidative status, and splenic inflammatory responses of broiler chickens under chronic heat stress, The Journal of Poultry Science, 2020). Therefore, ensuring the normal intestinal mucosal integrity and barrier function of poultry such as broilers is crucial to alleviate the negative effects caused by heat stress. Feed nutrition is an important aspect of alleviating heat stress in livestock and poultry that is simple, fast and economical. Zinc is an essential trace element for broilers, which is an essential component or activator of a variety of enzyme active functional proteins in the body, and is widely involved in a series of metabolic activities in the body (Vallee et al., The Biochemical basis of zinc physiology, Physiological Reviews, 1993; Yu et al., Organic zinc absorption by the intestine of broilers in vivo, British Journal of Nutrition, 2017).

[0003] The most commonly used form of zinc in livestock and poultry feed is inorganic zinc, such as zinc oxide or zinc sulfate, but the absorption and utilization rate of inorganic zinc in livestock and poultry is low (only 30-60%), and the zinc not absorbed by the body will be excreted with feces, causing environmental pollution, and easily destroying vitamins and other active substances in feed. Ashmead et al. (1993) suggested that organic trace elements with appropriate chelate strength can effectively prevent anti-nutritional factors or other factors affecting mineral element deposition or adsorption in the intestine after entering the digestive tract, which can effectively increase the amount of trace elements entering the body (Ashmead et al., Comparative intestinal absorption and subsequent metabolism of metal amino acid chelates and inorganic metal salts, The Roles of Amino Acid Chelates in Animal Nutrition, 1993). SUMMARY

[0004] The application provides the application of medium chelation strength protein zinc in improving the heat stress resistance of chickens, and proposes a new function of medium chelation strength protein zinc, uses medium chelation strength protein zinc as a new zinc source additive for additional zinc to improve the heat stress resistance of chickens, and has important theoretical and practical guiding significance for popularizing the use of medium chelation strength organic zinc, reducing environmental pollution and improving the heat stress resistance of broilers.

[0005] Technical scheme: In order to realize the above-mentioned purpose, the application of medium chelation strength protein zinc in improving the heat stress resistance of chickens.

[0006] Further, the application of medium chelation strength protein zinc in improving the small intestinal mucosal injury of heat stress chickens.

[0007] Further, the application of medium chelation strength protein zinc in preparing a feed additive for improving the heat stress resistance of chickens.

[0008] Further, the application of medium chelation strength protein zinc in preparing a feed additive for improving the small intestinal mucosal injury of heat stress chickens.

[0009] Further, the application of medium chelation strength protein zinc in preparing a feed additive for improving the small intestinal mucosal injury of heat stress chickens.

[0010] Further, the application of medium chelation strength protein zinc in preparing a feed additive for improving the small intestinal mucosal injury of heat stress chickens.

[0011] Further, the application of medium chelation strength protein zinc in preparing a feed additive for improving the small intestinal mucosal injury of heat stress chickens.

[0012] Further, the application of medium chelation strength protein zinc in preparing a feed additive for improving the small intestinal mucosal injury of heat stress chickens.

[0013] As a preferred, the medium chelation strength protein zinc is added to obtain a feed of 60mg Zn / kg.

[0014] The feed additive disclosed by the application comprises medium chelation strength protein zinc as a different supplement form of Zn 2+ supplement, and auxiliary materials required by the feed additive.

[0015] The present application first proposes the ability of medium chelating strength protein zinc in relieving the small intestinal mucosa injury of heat stress broilers. The present application uses the small intestinal mucosa injury in vivo model caused by heat stress to study the effects of adding inorganic or organic zinc on the mucosa integrity and barrier function of different small intestinal segments of broilers (villus height and crypt depth and their ratio, villus surface area, serum diamine oxidase activity, endotoxin and fluorescein isothiocyanate dextran (FITC-D). The present application successfully constructs the in vivo model of heat stress broilers, and the research results show that adding zinc can significantly reduce the increase of intestinal permeability and improve the damage of small intestinal barrier function of heat stress broilers induced by heat stress, and the addition of medium chelating strength protein zinc has more obvious relief effect than the addition of inorganic zinc sulfate, which fully shows that the addition of zinc in the feed, especially the addition of medium chelating strength protein zinc, can significantly improve the small intestinal mucosa injury of heat stress broilers. Medium chelating strength protein zinc can improve the increase of intestinal permeability and the destruction of small intestinal mucosa integrity caused by heat stress more than inorganic zinc sulfate, which reflects the ability of medium chelating strength protein zinc in improving the heat stress resistance of chickens.

[0016] The present application analyzes the correlation between medium chelating strength protein zinc and small intestinal mucosa injury of heat stress broilers by determining the effects of adding different zinc sources (inorganic zinc sulfate and medium chelating strength protein zinc) and zinc levels (30 and 60 mg / kg) on the small intestinal permeability and barrier function of heat stress broilers, so as to provide scientific experimental basis for the scientific application of medium chelating strength protein zinc in chicken production.

[0017] Advantages: Compared with the prior art, the present application has the following advantages:

[0018] The medium chelating strength protein zinc used in the present application is an economical and effective feed additive, and the present application proposes a new function of medium chelating strength protein zinc, which can relieve the small intestinal mucosa injury of heat stress broilers under heat stress conditions. Compared with the addition of inorganic zinc sulfate in the feed, the addition of medium chelating strength protein zinc in the feed has more obvious relief effect on the small intestinal mucosa injury of heat stress broilers, which is specifically manifested as the decrease of small intestinal permeability of broilers (reduction of FITC-D content, reduction of diamine oxidase activity, reduction of endotoxin content) and the enhancement of barrier function (reduction of crypt depth, increase of villus height, villus height / crypt depth, villus surface area and tight junction protein expression level). Therefore, the addition of medium chelating strength protein zinc to the feed with 60 mg Zn / kg is most beneficial to improve the small intestinal mucosa injury of heat stress broilers. The medium chelating strength protein zinc in the present application can be effectively used in the preparation of feed additives for improving the heat stress resistance of chickens, and the preparation of feed additives for improving the small intestinal mucosa injury of heat stress chickens. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 Effect of different zinc sources and zinc levels on serum FITC-D content, serum DAO activity of 28, 35 and 42-day-old broilers under heat stress and serum endotoxin content of 42-day-old broilers. A Serum FITC-D content of 28-day-old broilers. B Serum FITC-D content of 28-day-old broilers. C Serum FITC-D content of 35-day-old broilers. D Serum FITC-D content of 35-day-old broilers. E Serum FITC-D content of 42-day-old broilers. F Serum FITC-D content of 42-day-old broilers. G Serum DAO activity of 28-day-old broilers. H Serum DAO activity of 28-day-old broilers. I Serum DAO activity of 35-day-old broilers. J Serum DAO activity of 35-day-old broilers. K Serum DAO activity of 42-day-old broilers. L Serum DAO activity of 42-day-old broilers. M Serum endotoxin content of 42-day-old broilers. ZnS: inorganic zinc sulfate. Zn-Prot M: medium chelating strength protein zinc (Qf = 51.6). NT: normal temperature, 23 ± 1℃, 24 h / d. HT: high temperature, 9:00-17:00: 34 ± 1℃, 8 h / d, and 17:00-9:00: 28 ± 1℃. CON: control group. PF: pair group. Zinc was added on the basis of the control group. FITC-D: fluorescein isothiocyanate dextran. DAO: diamine oxidase. Values with different superscript letters are significantly different (P < 0.05). *P < 0.05. Data are means ± standard errors (n = 4-7).

[0020] Figure 2 Effect of different zinc sources and zinc levels on tissue morphology of small intestine of 28-day-old broilers under heat stress. A Villus height of duodenum. B Crypt depth of duodenum. C Villus height / crypt depth of duodenum. D Villus surface area of duodenum. E Villus height of jejunum. F Crypt depth of jejunum. G Villus height / crypt depth of jejunum. H Villus surface area of jejunum. I Villus height of ileum. J Crypt depth of ileum. K Villus height / crypt depth of ileum. L Villus surface area of ileum. ZnS: inorganic zinc sulfate. Zn-Prot M: medium chelating strength protein zinc (Qf = 51.6). NT: normal temperature, 23 ± 1℃, 24 h / d. HT: high temperature, 9:00-17:00: 34 ± 1℃, 8 h / d, and 17:00-9:00: 28 ± 1℃. CON: control group. PF: pair group. Zinc was added on the basis of the control group. Values with different superscript letters are significantly different (P < 0.05). *P < 0.05. Data are means ± standard errors (n = 5-7).

[0021] Figure 3 Effects of different zinc sources and zinc levels in diets on small intestine histomorphology of 35-day-old broilers under heat stress. A duodenum villus height. B duodenum crypt depth. C duodenum villus height / crypt depth. D duodenum villus surface area. E jejunum villus height. F jejunum crypt depth. G jejunum villus height / crypt depth. H jejunum villus surface area. I ileum villus height. J ileum crypt depth. K ileum villus height / crypt depth. L ileum villus surface area. ZnS: inorganic zinc sulfate. Zn-Prot M: medium chelating strength protein zinc (Qf = 51.6). NT: normal temperature, 23 ± 1℃, 24 h / d. HT: high temperature, 9:00-17:00: 34 ± 1℃, 8 h / d, and 17:00-9:00: 28 ± 1℃. CON: control group. PF: pair group. Zinc was added on the basis of the control group. Values with different superscripts differ (P < 0.05). *P < 0.05. Data are means ± standard errors (n = 5-7).

[0022] Figure 4 Effects of different zinc sources and zinc levels in diets on small intestine histomorphology of 42-day-old broilers under heat stress. A duodenum villus height. B duodenum crypt depth. C duodenum villus height / crypt depth. D duodenum villus surface area. E jejunum villus height. F jejunum crypt depth. G jejunum villus height / crypt depth. H jejunum villus surface area. I ileum villus height. J ileum crypt depth. K ileum villus height / crypt depth. L ileum villus surface area. ZnS: inorganic zinc sulfate. Zn-Prot M: medium chelating strength protein zinc (Qf = 51.6). NT: normal temperature, 23 ± 1℃, 24 h / d. HT: high temperature, 9:00-17:00: 34 ± 1℃, 8 h / d, and 17:00-9:00: 28 ± 1℃. CON: control group. PF: pair group. Zinc was added on the basis of the control group. Values with different superscripts differ (P < 0.05). *P < 0.05. Data are means ± standard errors (n = 4-7).

[0023] Figure 5Effect of different zinc sources and zinc levels in diet on jejunum PCNA positive cell number of 42-day-old broilers under heat stress. PCNA: proliferating cell nuclear antigen. ZnS: inorganic zinc sulfate. Zn-Prot M: medium chelating strength protein zinc (Qf = 51.6). NT: normal temperature, 23 ± 1 °C, 24 h / d. HT: high temperature, 9:00-17:00: 34 ± 1 °C, 8 h / d, and 17:00-9:00: 28 ± 1 °C. CON: control group. PF: pair group. Zinc was added on the basis of the control group. Values with different superscripts differ (P < 0.05). Data are means ± standard error (n = 6-7).

[0024] Figure 6 Effect of different zinc sources and zinc levels in diet on jejunum tight junction protein mRNA expression level of 42-day-old broilers under heat stress. Claudin-1: tight junction protein 1; occludin: occludin; JAM-A (junctional adhesion molecule A): junctional adhesion molecule A; ZO-1 (zona occludens 1): zona occludens 1. ZnS: inorganic zinc sulfate. Zn-Prot M: medium chelating strength protein zinc (Qf = 51.6). NT: normal temperature, 23 ± 1 °C, 24 h / d. HT: high temperature, 9:00-17:00: 34 ± 1 °C, 8 h / d, and 17:00-9:00: 28 ± 1 °C. CON: control group. PF: pair group. Zinc was added on the basis of the control group. Values with different superscripts differ (P < 0.05). Data are means ± standard error (n = 5-7).

[0025] Figure 7Effect of different zinc sources and zinc levels in diets on jejunal tight junction protein expression levels in 42-day-old broilers under heat stress. Claudin-1: tight junction protein 1; occludin: occludin; JAM-A (junctional adhesion molecule A): junctional adhesion molecule A; ZO-1 (zona occludens 1): zona occludens 1. ZnS: inorganic zinc sulfate. Zn-Prot M: medium chelating strength protein zinc (Qf = 51.6). NT: normal temperature, 23 ± 1℃, 24h / d. HT: high temperature, 9:00-17:00: 34 ± 1℃, 8h / d, and 17:00-9:00: 28 ± 1℃. CON: control group. PF: paired group. Zinc was added on the basis of the control group. Values with different superscripts differ significantly (P<0.05). *P<0.05. Data are means ± standard errors (n=5-7). DETAILED DESCRIPTION

[0026] The application will be further described below in connection with specific examples.

[0027] The raw materials used in the embodiments of the application are commercially available, or the same type of commercially available raw materials can be used.

[0028] The preparation method of medium chelating strength protein zinc (Qf = 51.6) is described in detail in the invention patent (Preparation method and use of a medium chelating strength protein zinc feed additive: 201610656482.5, Luo Xugang, 2016).

[0029] Example 1

[0030] One-day-old healthy AA broilers were purchased (1150) and all birds were allowed to freely access feed during 1-21 days of age. At 22 days of age, 784 birds were selected according to body weight and randomly divided into 7 treatment groups with 7 replicates per treatment group and 16 birds per replicate (2 adjacent cages, 8 birds per cage). Five treatment groups under heat stress and 2 treatment groups under normal temperature were placed in a large high-temperature chicken house and a small normal temperature chicken house, respectively, and the temperature in the house was controlled by a temperature control machine and a heat preservation lamp to be in a relatively stable range. The average feed intake of the paired groups was controlled to be consistent with the feed intake of the control group under heat stress, that is, the average feed intake of the control group on the previous day under heat stress was used as the average feed intake of the paired group on the second day. According to the NRC (1994) poultry nutritional requirements and the recommended nutritional requirements of meat chickens in China's chicken feeding standards (2004) as well as the recent research results of the laboratory on the zinc requirements of 1-21 day old (84 mg / kg) and 22-42 day old (65 mg / kg) meat chickens, 1-21 day old complete feed and 22-42 day old corn-soy meal type basic feed without added zinc were prepared (Table 1), and on the basis of the basic feed, inorganic zinc sulfate and medium chelation strength protein zinc were added to prepare the feed of each treatment group according to the following experimental design and treatment. In addition, synthetic methionine and lysine were added to balance the methionine and lysine levels in the feed of each treatment group. The feed was fed in the form of powder.

[0031] Experimental design and treatment:

[0032] This study was conducted using 22-day-old AA broiler roosters for a 21-day experimental period, from 22 to 42 days of age. A two-factor, completely randomized design was used: a heat-stressed control group (1 (zinc sulfate supplemented control group) + 2 (zinc sources) × 2 (zinc supplementation levels), for a total of five treatment groups; and a normothermic, single-factor, completely randomized design was used: a control group (zinc sulfate supplemented control group) + 1 (a pairing group whose feed intake was controlled to the same level as the heat-stressed control group), for a total of two treatment groups. Heat stress conditions were set at 34±1°C for 8 hours daily from 9:00 AM to 5:00 PM, with a temperature of 28±1°C during the remaining periods. Normothermal conditions were set at 23±1°C for 24 hours daily. Relative humidity was maintained at 55±5% during both heat-stress and normothermal conditions. Under heat stress conditions, two zinc sources were added: inorganic zinc sulfate and medium-chelated zinc protein. A control group, supplemented with zinc sulfate to meet the zinc nutritional requirement (65 mg / kg) for broilers aged 22-42 days, was used. Two dietary zinc supplementation levels were set at 30 and 60 mg / kg, respectively. Therefore, under heat stress conditions, there were five treatment groups. Under normal temperature conditions, one free-feeding control group meeting the zinc nutritional requirement (65 mg / kg) for broilers and one paired group to eliminate the effects of heat stress feed intake were set up. Therefore, this experiment had a total of seven treatment groups.

[0033] Table 1 Composition and nutritional level of broiler chicken diets (feeding basis)

[0034]

[0035]

[0036] a It is reagent grade.

[0037] b It is feed grade.

[0038] c Add VA 12000 IU, VD 34500 IU, VE 33 IU, VK 33 mg, VB 13 mg, VB 29.6 mg, VB 64.5 mg, VB 120.03 mg, Pantothenic acid calcium 15 mg, Niacin 54 mg, Folic acid 1.5 mg, Biotin 0.15 mg, Choline 700 mg, Cu (CuSO4 5H2O) 6 mg, Fe (FeSO4 7H2O) 40 mg, Zn (ZnSO4 7H2O) 60 mg, Mn (MnSO4 H2O) 110 mg, Se (Na2SeO3) 0.35 mg, I (Ca (IO3) 2 H2O) 0.35 mg. For broilers aged 22 to 42 days, add the following to each kilogram of feed: VA 8000 IU, VD 33000 IU, VE 22 IU, VK 32 mg, VB 12 mg, VB 26.4 mg, VB 63 mg, VB 12 0.02 mg, Pantothenic acid calcium10mg, Niacin 36mg, Folic acid 1.0mg, Biotin 0.10mg, Choline 500mg, Cu(CuSO4·5H2O)6mg, Fe(FeSO4·7H2O)30mg, Mn(MnSO4·H2O)80mg, Se(Na2SeO3)0.35mg, I(Ca(IO3)2·H2O)0.35mg.

[0039] d Zinc source was added by replacing an equal weight of corn starch. The treatment settings are detailed in Table 3.

[0040] e Calculated value.

[0041] f Measured value, each value is based on the average of the analysis results of three parallel samples

[0042] Example 2

[0043] 1. Sample collection and preparation

[0044] At the age of 28, 35 and 42 days of the experiment of Example 1, after the chickens were deprived of food but not water for one night, 3 chickens close to the average body weight were selected from each replicate, and 5 mL of blood was collected from the wing vein and hepatic portal vein of each chicken using a disposable blood collection needle into a non-anticoagulant vacuum tube. After centrifugation at 3000 r / min for 10 min, the serum was collected and stored at -20℃ for the determination of endotoxin and diamine oxidase activity in the serum. Then the chickens were slaughtered, and the small intestine of one of the chickens was removed, and the duodenum, jejunum and ileum segments were isolated, and 2 cm of the middle part of each small intestine segment was cut and placed in a 4% paraformaldehyde solution for analysis of the morphology of each small intestine segment (villus height, crypt depth and their ratio, and villus surface area) and the number of PCNA positive cells in the jejunum. The remaining intestinal segment after the jejunum of the first chicken and the jejunum of the remaining two chickens were washed with ice-cold physiological saline to remove the intestinal contents, and then the intestinal mucosa was scraped with a sterile glass slide, immediately placed in a sterile 1.5 mL centrifuge tube, and stored at -80℃ after quick freezing in liquid nitrogen for detection of tight junction protein [Claudin-1, Occludin, Zonula occluden (ZO)-1, Junctional adhesion molecule (JAM)-A] mRNA and its translated protein expression levels. Another 1 chicken close to the average body weight was selected from each replicate, and was orally administered with FITC-D solution (purchased from Sigma-Aldrich MO, item number: FD4) at a dose of 8.32 mg FITC-D / kg body weight. Specifically, the FITC-D was dissolved in 0.9% physiological saline to prepare a solution containing 8.32 mg / mL of FITC-D, and then the required amount of FITC-D solution was calculated according to the body weight of the chickens in each treatment group, and 5 mL of the solution was injected into the oral cavity of each chicken through the throat using a 5 mL pipette, and it was ensured that all the administered solution was not lost. After 1 h, 5 mL of blood was collected from the wing vein and hepatic portal vein of the chicken using a disposable blood collection needle into a non-anticoagulant vacuum tube, and after centrifugation at 3000 r / min for 10 min, the serum was collected and stored at -20℃ for detection of serum FITC-D content (Zhang et al., 2021, Colon tissue-accumulating mesoporous carbon nanoparticles loaded with musca domesticacecropin for ulcerative colitis therapy, Theranostics), and the chicken was slaughtered after blood collection. For samples of 3 chickens in each replicate, the samples of the 3 chickens were combined in equal proportions as one sample for analysis.

[0045] 2. Sample analysis

[0046] The fixed tissues were stained with hematoxylin and eosin, observed under a microscope, and morphological indicators were determined using an image processing and analysis system (Guangzhou Ming Mei Technology Co., Ltd. China). The measurement indicators included villus height, crypt depth, and villus width. Three typical fields (villus complete and straight) were selected for each section, and the longest villus height and corresponding crypt depth and villus width were selected for each field. The average values were taken as the measurement results. Villus surface area = 2πrh, where r is the radius (half of the villus width) and h is the villus height (Wu et al., 2017). Fluorescence was measured using a Synergy HT multi-mode microplate reader (Biotek, Winooski, VT) with a dual-wavelength multifunctional enzyme labeler at an excitation wavelength of 428 nm and an emission wavelength of 528 nm to detect serum FITC-D content (Tabler et al., 2020, Intestinal barrier integrity in heat-stressed modern broilers and their ancestor wild jungle fowl, Frontiers In Veterinary Science). Proliferating cell nuclear antigen (PCNA) immunohistochemical staining was performed on paraffin-embedded tissue sections. PCNA-positive cells were brown in the nucleus, and the number of brown-stained cells in the nucleus was recorded as the number of PCNA-positive cells. The mRNA and translated protein expression levels of tight junction proteins (Claudin-1, Occludin, ZO-1, JAM-A) in the jejunum mucosa were determined by SYBR Green RT-qPCR and Western blotting, respectively.

[0047] 3. Statistical analysis

[0048] Statistical analysis of the experimental data was performed using SAS 9.4 (2013). The significance of differences between the heat stress control group and the zinc supplementation groups (30 and 60 mg / kg) was tested using the single degree of freedom comparison method (Liu et al., 2020, Relative bioavailability of selenium yeast for broilers fed a conventional corn-soybean meal diet, Journal of Animal Physiology and Animal Nutrition). A 2×2 two-way analysis of variance (ANOVA) was performed on all data except the heat stress control group using the general linear model (GLM) procedure, with the statistical model including zinc source, zinc supplementation level, and the interaction between the two. A one-way ANOVA was also performed on the heat stress control group and the ad libitum control and paired groups under normal temperature. For significant differences in the ANOVA, the least significant difference (LSD) method was used to compare the differences between the means. Each replicate was considered an experimental unit. A P < 0.05 level of significance was used to test for differences.

[0049] 4. Results

[0050] 4.1 Effects of different zinc sources and zinc levels in the diet on FITC-D content and diamine oxidase activity in serum of 28-, 35-, and 42-day-old broilers under heat stress conditions, as well as serum endotoxin content in 42-day-old broilers

[0051] The results are as follows Figure 1 .Depend on Figure 1 Heat stress significantly increased (P < 0.05) the FITC-D content and diamine oxidase activity in the wing vein serum and the portal vein serum at 35 and 42 days of age in broiler chickens. It also significantly increased (P < 0.05) the diamine oxidase activity in the portal vein serum and the endotoxin content in the portal vein serum at 42 days of age. As shown in Table 2, compared with dietary Zn supplementation at 30 mg / kg, high-level Zn supplementation (60 mg / kg) significantly reduced (P < 0.05) the FITC-D content and diamine oxidase activity in the wing vein serum at 42 days of age. A comparison of the sum of dietary inorganic Zn at two Zn supplementation levels (30 and 60 mg / kg) with the sum of dietary organic Zn with medium chelation strength at two Zn supplementation levels (30 and 60 mg / kg) showed that dietary protein Zn supplementation with medium chelation strength significantly reduced (P < 0.05) the endotoxin content in the portal vein serum at 42 days of age compared with dietary zinc sulfate supplementation.

[0052] 4.2 Effects of dietary zinc supplementation with different zinc sources and levels on the histomorphology of small intestine segments in broiler chickens at 28, 35, and 42 days of age under heat stress

[0053] The results are as follows Figure 2 、 Figure 3 and Figure 4 .Depend on Figure 2 、 Figure 3 and Figure 4 It can be seen that heat stress significantly increased (P<0.05) the crypt depth of each segment of the small intestine (duodenum, jejunum and ileum) of broiler chickens at 28, 35 and 42 days of age, and significantly decreased (P<0.05) the villus height (except the duodenum and ileum at 28 days of age), villus height / crypt depth and villus surface area (except the ileum at 28 days of age) of each segment of the small intestine of broiler chickens at 28, 35 and 42 days of age. Compared with the addition of inorganic zinc sulfate in the diet, the addition of medium-chelated strength protein zinc in the diet significantly reduced (P<0.05) the crypt depth of the jejunum of 28-day-old broiler chickens, the duodenum of 35-day-old broiler chickens, and the ileum of 42-day-old broiler chickens; significantly increased (P<0.05) the villus height of the jejunum and ileum of 28-day-old broiler chickens, the duodenum and jejunum of 35-day-old broiler chickens, and the ileum of 42-day-old broiler chickens; significantly increased (P<0.05) the villus height / crypt depth of the ileum of 28-day-old broiler chickens, the duodenum, jejunum and ileum of 35-day-old broiler chickens, and the jejunum and ileum of 42-day-old broiler chickens; and significantly increased (P<0.05) the villus surface area of ​​the duodenum, jejunum and ileum of 28-day-old broiler chickens, the ileum of 35-day-old broiler chickens, and the duodenum and jejunum of 42-day-old broiler chickens.

[0054] Compared with the diet supplemented with 30 mg / kg zinc, the diet supplemented with 60 mg / kg zinc (on the basis of meeting the zinc requirement) significantly reduced (P<0.05) the crypt depth of the jejunum and ileum of 28-day-old broiler chickens, the duodenum of 35-day-old broiler chickens and the ileum of 42-day-old broiler chickens (P<0.05); significantly increased (P<0.05) the villus height of the duodenum and jejunum of 42-day-old broiler chickens; significantly increased (P<0.05) the villus height / crypt depth of the ileum of 28-day-old broiler chickens, the jejunum and ileum of 42-day-old broiler chickens; and significantly increased (P<0.05) the villus surface area of ​​the jejunum of 42-day-old broiler chickens.

[0055] 4.3 Effects of dietary zinc supplementation with different zinc sources and zinc levels on the number of PCNA-positive cells in the jejunum of 42-day-old broiler chickens under heat stress

[0056] The results are as follows Figure 5 .Depend on Figure 5Heat stress significantly increased (P<0.05) the number of PCNA-positive cells in the jejunum of 42-day-old broiler chickens. Compared with dietary supplementation with inorganic zinc sulfate, dietary supplementation with medium-chelated protein zinc significantly increased (P<0.05) the number of PCNA-positive cells in the jejunum of 42-day-old broiler chickens. Compared with dietary supplementation with 30 mg / kg zinc, dietary supplementation with 60 mg / kg zinc (on the basis of meeting zinc requirements) significantly increased the number of PCNA-positive cells in the jejunum of 42-day-old broiler chickens.

[0057] 4.4 Effects of dietary zinc supplementation with different zinc sources and zinc levels on the expression of tight junction proteins mRNA and protein in the jejunum of 42-day-old broiler chickens under heat stress

[0058] The results are as follows Figure 6 and 7 .Depend on Figure 6 and 7 Heat stress significantly decreased (P < 0.05) the mRNA and protein expression levels of Claudin-1, Occludin, ZO-1, and JAMA. Compared with dietary zinc sulfate supplementation, dietary supplementation with medium-chelated protein zinc significantly increased (P < 0.05) the protein expression levels of Claudin-1, Occludin, and JAMA in the jejunum of 42-day-old broilers. Compared with dietary zinc supplementation at 30 mg / kg, dietary zinc supplementation at 60 mg / kg (on top of meeting zinc requirements) significantly increased (P < 0.05) the mRNA expression levels of Claudin-1 and ZO-1 and the protein expression levels of Claudin-1, Occludin, and JAMA in the jejunum of 42-day-old broilers.

[0059] 5. Conclusion

[0060] Zinc supplementation under heat stress conditions can alleviate small intestinal mucosal damage in heat-stressed broiler chickens. The moderately chelated organic zinc group (60 mg Zn / kg) showed the most significant mitigating effect on small intestinal mucosal damage in heat-stressed broiler chickens. This was demonstrated by decreased permeability in various segments of the small intestine (reduced FITC-D content, decreased diamine oxidase activity, and decreased endotoxin content) and enhanced barrier function (reduced crypt depth, increased villus height, villus height / crypt depth, and villus surface area, and increased tight junction protein expression). Therefore, dietary supplementation with moderately chelated protein zinc is most beneficial for improving small intestinal mucosal damage in heat-stressed broiler chickens.

Claims

1. Use of medium-strength protein zinc chelator in improving the heat stress resistance of chickens.

2. Use of medium-strength protein zinc chelator in improving the small intestinal mucosal injury of heat-stressed chickens.

3. Use of medium-strength protein zinc chelator in preparing a feed additive for improving the heat stress resistance of chickens.

4. Use of medium-strength protein zinc chelator in preparing a feed additive for improving the small intestinal mucosal injury of heat-stressed chickens.

5. Use according to any one of claims 1 to 4, characterized in that, The use of the medium-strength protein zinc chelator as a feed additive on the basis of animal feed in improving the heat stress resistance of chickens.

6. Use according to claim 5, characterized in that, The animal feed is broiler feed.

7. Use according to claim 6, characterized in that, The medium chelating strength protein zinc is added in the feed as Zn 2 + supplement.

8. Use according to claim 6, characterized in that, The medium-strength protein zinc chelator is preferably added to the feed to obtain a feed of 30-60 mg Zn / kg.

9. A feed additive for improving the heat stress tolerance of chickens, characterized in that, The feed additive comprises the medium-strength protein zinc chelator as an effective component, and auxiliary materials required for the feed additive.

10. A feed additive for ameliorating small intestinal mucosal lesion in heat-stressed chicken, characterized by comprising: The feed additive includes medium chelating strength protein zinc as different supplementary forms of Zn 2+ Supplements and auxiliary materials required for feed additives.

Images