3-hydroxyanthranilic acid improves the production performance of laying hens in the late laying period

Abstract

This invention discloses the application of 3-hydroxy-2-aminobenzoic acid (3-hydroxy-2-aminobenzoic acid) in improving the laying performance of hens in the late laying period, relating to the field of animal nutrition and feed additive technology. This invention provides the application of 3-hydroxy-2-aminobenzoic acid in the preparation of feed additives or feeds for improving the laying performance of hens in the late laying period. Adding 3-hydroxy-2-aminobenzoic acid with a purity of not less than 95% to a complete diet of laying hens at a dosage of 75-600 mg / kg, with a preferred dosage of 300 mg / kg, significantly improves egg production rate, reduces feed conversion ratio, and improves egg quality; it also improves reproductive phenotype, increases serum reproductive hormone levels, and increases the number of effective follicles; and it exerts its effects by enhancing ovarian antioxidant capacity and regulating the expression of inflammatory factors and related genes. This invention provides a new, efficient, and stable feed additive option for improving the laying performance of hens in the late laying period.

Description

Technical Field

[0001] This invention relates to the field of animal nutrition and feed additives, and in particular to the application of 3-hydroxy-o-aminobenzoic acid in improving the laying performance of laying hens in the later stages of egg production. Background Technology

[0002] As laying hens enter the late laying period, egg production typically declines gradually, the feed conversion ratio increases, and eggshell strength and internal egg quality fluctuate. This is accompanied by changes in follicle recruitment, follicle maturation, and local ovarian homeostasis. Extending the efficient laying cycle can reduce flock turnover and increase feed efficiency per unit area. Therefore, developing feed active ingredients suitable for late-laying hens with clearly defined dosages and repeatable effects has practical production value.

[0003] Currently, nutritional measures used to maintain late-laying egg production performance mostly focus on vitamins, trace elements, plant extracts, or compound additives. However, the varying batches of raw materials and the complexity of their components can lead to differences in effectiveness. Furthermore, some protocols primarily observe production performance, lacking validation from multiple perspectives, including follicle count, reproductive hormones, local ovarian oxidation and inflammation, and yolk precursor utilization.

[0004] 3-Hydroxy-annaminobenzoic acid, also known as 2-amino-3-hydroxybenzoic acid or 3-hydroxyanthranilic acid, is a small molecule metabolite in the tryptophan-kynurenine metabolic pathway. Studies suggest that this molecule possesses redox and immunomodulatory biological activities, but no indication has been found that it improves egg production performance in the later stages of egg production in laying hens.

[0005] Therefore, there is a need to develop efficient and stable feed additives to improve the egg production performance of laying hens in the later stages of egg production. Summary of the Invention

[0006] The purpose of this invention is to provide an application of 3-hydroxy-o-aminobenzoic acid (3-hydroxy-o-aminobenzoic acid) to improve the laying performance of hens in the late laying period, thereby solving the problems existing in the prior art. This invention aims to provide a new use for 3-hydroxy-o-aminobenzoic acid in the feed of laying hens in the late laying period, addressing the problems of decreased egg production rate, increased feed conversion ratio, and weakened follicle development in the late laying period, and determining the optimal range and dosage of dietary addition to achieve stable production results.

[0007] To achieve the above objectives, the present invention provides the following solution: This invention provides the application of 3-hydroxy-o-aminobenzoic acid in the preparation of feed additives or feeds for improving the egg production performance of laying hens in the late laying period, wherein the purity of 3-hydroxy-o-aminobenzoic acid is not less than 95%.

[0008] Furthermore, the addition amount of 3-hydroxy-o-aminobenzoic acid in the complete diet of laying hens is 75-600 mg / kg.

[0009] Furthermore, the addition amount of 3-hydroxy-an-aminobenzoic acid in the complete diet of laying hens is 150-300 mg / kg.

[0010] Furthermore, the addition amount of 3-hydroxy-o-aminobenzoic acid in the complete diet of laying hens is 300 mg / kg.

[0011] Furthermore, improving egg production performance includes increasing the egg production rate and / or reducing the feed conversion ratio.

[0012] Furthermore, the application also includes improving egg quality, which includes increasing at least one of eggshell strength, albumen height, and Haugh units.

[0013] Furthermore, improved egg production performance is accompanied by improved reproductive phenotype; improved reproductive phenotype includes at least one of the following: increased serum follicle-stimulating hormone, luteinizing hormone, progesterone or estradiol levels, increased number of small follicles, large white follicles or total follicles, and / or decreased number of atretic follicles.

[0014] Furthermore, improved egg production performance is accompanied by increased total antioxidant capacity or superoxide dismutase activity in the ovary, decreased malondialdehyde levels in the ovary, decreased levels of tumor necrosis factor-α or interleukin-1β in the ovary, and / or increased levels of interleukin-10 in the ovary.

[0015] The present invention also provides a feed additive for improving the egg production performance of laying hens in the later stage of egg production. The feed additive contains 3-hydroxy-o-aminobenzoic acid and a feed carrier. After the feed additive is added to the complete diet, the content of 3-hydroxy-o-aminobenzoic acid in the complete diet is 75-600 mg / kg.

[0016] The present invention also provides a late-laying hen feed, which includes a basal diet and 3-hydroxy-2-aminobenzoic acid, and contains 75-600 mg of 3-hydroxy-2-aminobenzoic acid per kilogram of feed.

[0017] 3-Hydroxy-o-aminobenzoic acid can be directly premixed with a small amount of basal feed and then added to a complete diet. Alternatively, it can be mixed with rice bran, corn cob powder, silica, or other feed carriers to form a premixed feed additive before being added to the complete diet. The dosage of 3-hydroxy-o-aminobenzoic acid in this invention is based on its formulation in a complete diet and is not limited by the type of carrier or the premixing ratio.

[0018] 3-Hydroxy-o-aminobenzoic acid is preferably prepared with a purity of not less than 95%. The remaining nutritional components of the feed can be formulated according to the nutritional requirements of the laying hen breed, age, and production stage.

[0019] The present invention discloses the following technical effects: Compared with basal diets without added 3-hydroxy-o-aminobenzoic acid, this invention improved egg production rate and reduced feed conversion ratio within the tested range of 75-600 mg / kg. Specifically, in the 300 mg / kg group, the total egg production rate increased from 69.79% to 87.84%, an increase of 18.05 percentage points, or a relative increase of 25.86%; the feed conversion ratio decreased from 2.18 to 2.08, a relative decrease of 4.59%.

[0020] The 300 mg / kg group simultaneously improved eggshell strength, albumen height, and Hardy units; increased serum follicle-stimulating hormone, luteinizing hormone, progesterone, and estradiol levels; increased the number of small follicles, large white follicles, and total follicles; and reduced the number of atretic follicles. Further ovarian assays showed that the appropriate dose could improve total antioxidant capacity and superoxide dismutase activity, reduce malondialdehyde levels, and have a beneficial regulatory effect on tumor necrosis factor-α, interleukin-1β, and interleukin-10.

[0021] Molecular detection results showed that the effects of this technology were accompanied by upregulation of the expression of liver APOVLDL2 and APOB, ovarian VLDLR and some steroid production-related genes, as well as an increase in the ovarian BCL2 / BAX ratio. These results support the correlation between "yolk precursor supply and uptake—follicle development—egg production performance," but this invention does not rely on a single signaling pathway as a necessary condition for achieving the technical effects.

[0022] The present invention observed that the overall effect of the moderate dose was better than that of the highest experimental dose, suggesting that the dose-response relationship is not a simple linear one. Therefore, 300 mg / kg was determined as the preferred dose in this embodiment. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 Representative images of the ovaries and follicles of laying hens in the late laying period after treatment with different doses of 3-hydroxy-o-aminobenzoic acid; Figure 2 Effects of different doses of 3-hydroxy-o-aminobenzoic acid on the liver of laying hens VTG2 , APOVLDL2 , MTTP and APOB The effect of relative expression levels; data in the figure are expressed as mean ± SEM. Compared with the control group, * indicates P<0.05, ** indicates P<0.01, and *** indicates P<0.001. Figure 3 The effects of different doses of 3-hydroxy-o-aminobenzoic acid on the ovaries of laying hens VLDLR , FSHR , LHCGR , CYP11A1 and CYP19A 1. Effect of relative expression levels; Data in the figure are expressed as mean ± SEM. Compared with the control group, * indicates P<0.05, ** indicates P<0.01, and *** indicates P<0.001. Figure 4 The effects of different doses of 3-hydroxy-o-aminobenzoic acid on the ovaries of laying hens BAX, BCL2, CASP3 Effects of relative expression levels and BCL2 / BAX ratio; data in the figure are expressed as mean ± SEM. Compared with the control group, * indicates P<0.05, ** indicates P<0.01, and *** indicates P<0.001. Detailed Implementation

[0025] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0026] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0027] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0028] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0029] The terms “include,” “including,” “have,” and “contain” used in this article are all open-ended terms, meaning that they include but are not limited to.

[0030] 3-Hydroxyanthranilic acid is 2-amino-3-hydroxybenzoic acid, with the English name 3-Hydroxyanthranilic acid, abbreviated as 3-HAA, and the CAS number 548-93-6.

[0031] Example 1 3-Hydroxy-o-aminobenzoic acid feeding trial Experimental Methods: Two hundred and forty commercial laying hens, approximately 500 days old, in good health, and with similar initial egg production rates, were randomly divided into five treatment groups. Each treatment had eight biological replicates, with six hens per replicate. The number of hens and rearing conditions were consistent across replicates. The control group was fed a basal diet. Experimental groups 1, 2, 3, and 4 were fed their basal diets supplemented with 75, 150, 300, and 600 mg / kg of 3-hydroxy-o-aminobenzoic acid, respectively. The purity of the experimental substance was not less than 95%.

[0032] 3-hydroxy-o-aminobenzoic acid was first premixed thoroughly with a small amount of basal diet, and then gradually integrated into the complete diet of each group using a step-by-step expansion method to ensure homogeneity. During the experiment, all groups were fed at the same stocking density, temperature, light, water, and immunization management, with free access to feed and water, for 10 consecutive weeks. Performance was statistically analyzed using replicates, recording daily egg production, total egg weight, feed intake, and abnormal egg occurrences, and calculating the egg production rate and feed conversion ratio.

[0033] At the end of the experiment, representative egg samples were randomly selected from each treatment group in replicates to determine eggshell strength, eggshell thickness, albumen height, Haugh units, yolk weight, and yolk color. Serum and tissue parameters were measured using independent biological samples. Data in the table are expressed as mean and standard error of mean (SEM). One-way ANOVA was used to compare treatment effects, and multiple comparisons between groups were performed; different superscript letters in the same row indicate significant differences (…). P <0.05).

[0034] Table 1. Effects of 3-hydroxy-o-aminobenzoic acid on egg production performance and egg quality.

[0035] Note: Data in the same row with no identical letters in the subscripts indicates a significant difference (P<0.05); items without subscripts indicate no significant difference between groups. Eggshell thickness increased from the original 10 -2 Convert mm measurement values ​​to mm.

[0036] As shown in Table 1, compared with the control group, the egg production rate of all four supplementary groups was significantly increased, and the feed conversion ratio was significantly decreased. The 300 mg / kg group had the highest egg production rate, and eggshell strength, albumen height, and Haugh units also reached high levels. There were no significant differences in average egg weight and average daily feed intake among the groups, indicating that the improvement in egg production rate was not due to a significant increase in feed intake. There were no significant differences in eggshell thickness, yolk weight, and yolk color among the groups; therefore, these are not considered as the main technical effects of this invention.

[0037] Example 2 Reproductive hormone and follicle development testing Detection methods: Serum was collected at the end of the experiment, and follicle-stimulating hormone (FSH), luteinizing hormone (LH), progesterone, and estradiol were measured using a chicken-specific enzyme-linked immunosorbent assay (ELISA) kit. After necropsy, small follicles, large white follicles, small yellow follicles, graded follicles, and atretic follicles were counted according to a unified judgment standard, and the total number of follicles was calculated. The results are shown in Tables 2 and 3, and the representative appearance of the ovary and follicles is shown in Tables 2 and 3. Figure 1 .

[0038] Table 2 Effects of 3-hydroxy-o-aminobenzoic acid on serum reproductive hormones

[0039] Note: Data in the same row with no identical letters in the superscript indicates a significant difference (P<0.05).

[0040] Table 3. Effect of 3-hydroxy-o-aminobenzoic acid on follicle number

[0041] Note: Data in the same row with no identical letters under the headings indicates a significant difference (P<0.05); items without headings indicate no significant difference between groups.

[0042] As shown in Tables 2 and 3, the 300 mg / kg group had the highest levels of follicle-stimulating hormone (FSH), progesterone, and estradiol, and its luteinizing hormone (LH) levels were also significantly higher than those in the control group. Compared with the control group, the number of small follicles, large white follicles, and total follicles increased by 25.41%, 24.59%, and 26.38%, respectively, while the number of atretic follicles decreased by 62.50%. Although the numbers of small yellow follicles and graded follicles increased, these increases did not reach statistical significance.

[0043] Example 3 Ovarian antioxidant and inflammatory status Experimental methods: Equal volumes of ovarian tissue were weighed and homogenized, and the supernatant was collected after centrifugation. Total antioxidant capacity, superoxide dismutase activity, glutathione peroxidase activity, and malondialdehyde content were determined using biochemical reagent kits. Tumor necrosis factor-α, interleukin-1β, interleukin-6, and interleukin-10 were determined using a chicken-specific enzyme-linked immunosorbent assay (ELISA) kit. Tissue results were normalized to total protein. Results are shown in Tables 4 and 5.

[0044] Table 4. Effects of 3-hydroxy-o-aminobenzoic acid on ovarian antioxidant indices

[0045] Note: Data in the same row with no identical letters in the superscript indicates a significant difference (P<0.05); there was no significant difference between the GSH-Px groups.

[0046] Table 5. Effects of 3-hydroxy-o-aminobenzoic acid on ovarian inflammatory factors

[0047] Note: Data in the same row with no identical letters in the superscript indicates a significant difference (P<0.05); there was no significant difference in IL-6 between groups.

[0048] As shown in Table 4, the total antioxidant capacity of the ovaries in the 150 and 300 mg / kg groups was significantly higher than that in the control group; the superoxide dismutase activity in the 300 mg / kg group increased by 96.92%, and malondialdehyde decreased by 27.69%. No significant difference was found in glutathione peroxidase.

[0049] As shown in Table 5, the levels of tumor necrosis factor-α and interleukin-1β were lower in the 150 and 300 mg / kg groups, while the level of interleukin-10 was significantly higher; interleukin-6 did not show significant changes. Therefore, this example supports the beneficial regulation of some ovarian inflammatory factors.

[0050] Example 4 Vitellogenin utilization, follicle function, and expression of apoptosis-related genes Experimental Methods: Total RNA was extracted from liver and ovarian tissues, reverse transcribed, and then subjected to real-time quantitative PCR. Normalization was performed using a suitable internal control gene, with the relative expression level of the control group set as 1. Eight independent biological samples were used in each group. Total RNA was extracted from liver and ovarian tissues using FreeZol™ reagent (R711-01, Novizan Biosciences Co., Ltd.) according to the kit instructions. RNA concentration and purity were detected using a NanoDrop micro-spectrophotometer. The total RNA was then reverse transcribed into cDNA using the HiScript III first-strand cDNA synthesis kit (R312-01, Novizan Biosciences Co., Ltd.). Amplification was performed using a Taq Pro Universal SYBR qPCR Master Mix (Q712-02, Novizan Biosciences Co., Ltd.) on a CFX Connect™ real-time quantitative PCR detection system (Bio-Rad Laboratories, USA). The total volume of each reaction was 20 μL, including 10 μL qPCR Master Mix, 1 μL cDNA template, 0.4 μL forward primer (10 μmol / L), 0.4 μL reverse primer (10 μmol / L), and 8.2 μL nuclease-free water. The amplification program was: 95℃ pre-denaturation for 30 s; followed by 40 cycles, each consisting of 95℃ denaturation for 5 s, 60℃ annealing for 30 s, and 72℃ extension for 30 s. The primer sequences used are shown in Table 6. β-actin was used as an internal reference gene, and 2... -ΔΔCt The relative mRNA expression level of the target gene was calculated using the method. Data in the figure are expressed as mean ± SEM. Compared with the control group, * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001.

[0051] like Figure 2 As shown, the livers of the 150 and 300 mg / kg groups APOVLDL2 The relative expression level was significantly increased in the 300 and 600 mg / kg groups. APOB The relative expression level increased significantly; VTG2 and MTTP No significant changes were observed. This result suggests that 3-hydroxy-anaminobenzoic acid selectively regulates yolk precursor-related genes.

[0052] like Figure 3 As shown, the ovaries in the 150 and 300 mg / kg groups VLDLR The relative expression level was significantly increased in the 75, 300, and 600 mg / kg groups. CYP11A1 The relative expression level was significantly increased; in the 300 mg / kg group CYP19A1 The relative expression level increased significantly. FSHR and LHCGRNo significant changes were observed. The above changes are consistent with the improvement in serum reproductive hormones and follicle count.

[0053] like Figure 4 As shown, the 300mg / kg group BAX The relative expression level was significantly reduced. BCL2 The relative expression level was significantly increased; the BCL2 / BAX ratio was increased in all addition groups, with the highest value in the 300 mg / kg group. CASP3 No significant change was observed in relative expression levels. Therefore, the results support a shift in the apoptosis regulatory balance towards anti-apoptosis.

[0054] Table 6 Primer sequences used for real-time quantitative PCR (qRT-PCR) detection

[0055] Example 5 Premixed additives and their usage Method: To improve the uniformity of mixing small doses of active ingredients in large batches of feed, 3-hydroxy-an-aminobenzoic acid can be formulated into a premixed additive with a feed carrier. For example, when preparing a premix containing 30% (mass percentage) 3-hydroxy-an-aminobenzoic acid, 300g of 3-hydroxy-an-aminobenzoic acid can be mixed stepwise with 700g of feed carrier; adding 1kg of this premix to each ton of complete feed will bring the 3-hydroxy-an-aminobenzoic acid content in the complete feed to 300mg / kg. This example only changes the dispersion and feeding method, and does not change the final concentration of the active ingredient verified in Example 1.

[0056] When the target dosage is 75, 150, or 600 mg / kg, the proportion of active ingredients in the premix or the amount of premix added can be adjusted accordingly. The premix should be protected from light, sealed, and stored according to the raw material stability requirements; its production and application should also comply with the relevant regulatory requirements for feed and feed additives.

[0057] In summary, 3-hydroxy-annaminobenzoic acid (3-hydroxy-2-aminobenzoic acid) supplemented in the diet at a concentration of 75-600 mg / kg improved egg production and feed conversion ratio in late-laying hens. The 300 mg / kg concentration showed the most comprehensive beneficial effects in terms of production performance, egg quality, reproductive hormones, follicle count, and local ovarian condition. The 150 mg / kg concentration showed outstanding performance on some inflammatory markers, and the 600 mg / kg concentration retained some production benefits, but its overall performance did not exceed that of the 300 mg / kg concentration. This further illustrates that the optimal dosage should be determined based on multiple indicators rather than a single endpoint.

[0058] In the application of the 3-hydroxy-o-aminobenzoic acid of this invention, the average daily feed intake and average egg weight of chickens did not show any significant adverse changes.

[0059] Example 6 Safety test Forty-eight healthy late-laying hens were randomly divided into a control group and an experimental group, with eight replicates per group and three hens per replicate. The control group was fed a basal diet, while the experimental group was fed a diet mixed with feed at five times the recommended dosage (300 mg / kg) for 14 consecutive days. During the experiment, the mental state, activity, feed intake, water intake, and fecal characteristics of the hens in all groups were normal, and no deaths or other obvious abnormalities occurred. After the experiment, necropsy was performed, and no abnormal lesions were found in the major organs.

[0060] The results showed that under the experimental conditions, feeding the 3-hydroxy-o-aminobenzoic acid of this invention at 5 times the recommended addition amount for 14 consecutive days did not cause any obvious adverse reactions in laying hens, indicating that its application in laying hen feed is safe.

[0061] The actual application of the feed additives described in this invention can only be implemented after approval by the relevant competent authorities in accordance with local feed regulations.

[0062] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

The application of 1,3-hydroxy-o-aminobenzoic acid in the preparation of feed additives or feeds for improving follicle development and enhancing egg production performance in late-laying hens, characterized in that... The purity of the 3-hydroxy-o-aminobenzoic acid is not less than 95%; the addition amount of the 3-hydroxy-o-aminobenzoic acid in the complete diet of laying hens is 75-600 mg / kg.

2. The application according to claim 1, characterized in that, The addition amount of 3-hydroxy-o-aminobenzoic acid in the complete diet of laying hens is 150-300 mg / kg.

3. The application according to claim 2, characterized in that, The addition amount of 3-hydroxy-o-aminobenzoic acid in the complete diet of laying hens is 300 mg / kg.

4. The application according to any one of claims 1-3, characterized in that, Improving egg production performance includes increasing the egg production rate and / or reducing the feed conversion ratio.

5. The application according to claim 4, characterized in that, The application also includes improving egg quality, which includes increasing at least one of eggshell strength, albumen height, and Haugh units.

6. The application according to claim 1, characterized in that, The improvement of follicle development in late-laying hens includes at least one of increasing the number of small follicles, large white follicles, or total follicles, and / or decreasing the number of atretic follicles.

7. The application according to claim 6, characterized in that, The 3-hydroxy-o-aminobenzoic acid improves follicle development by increasing at least one of serum follicle-stimulating hormone, luteinizing hormone, progesterone, or estradiol levels.

8. A feed additive for improving follicle development and enhancing egg production performance in laying hens during the later stages of egg production, characterized in that, The feed additive contains 3-hydroxy-2-aminobenzoic acid and a feed carrier; when the feed additive is added to a complete diet, the content of 3-hydroxy-2-aminobenzoic acid in the complete diet is 75-600 mg / kg.

9. A feed for improving follicle development and enhancing egg production performance in laying hens during the later stages of egg production, characterized in that, The feed comprises a basal diet and 3-hydroxy-2-aminobenzoic acid, and contains 75-600 mg of 3-hydroxy-2-aminobenzoic acid per kilogram of the feed.

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