A feed additive composition containing kaempferol and curcumin and its application in relieving heat stress of laying hens

Abstract

The application provides a feed additive composition containing kaempferol and curcumin and application thereof in relieving heat stress of laying hens, and belongs to the technical field of feed additives.The active ingredient of the feed additive composition is composed of kaempferol and curcumin in a mass ratio of 1:2, and the addition amount in the feed is 600 mg / kg.The application realizes bidirectional positive feedback amplification of antioxidation and anti-inflammation through the synergistic interaction of kaempferol in activating the Nrf2 / ARE antioxidation pathway and curcumin in inhibiting the NF-kappa B inflammation pathway, and synergistically regulates the hypothalamus-pituitary-adrenal axis and the hypothalamus-pituitary-gonad axis, so as to reduce the serum corticosterone level and improve the estradiol level.The application systematically solves the technical problems of single action target and unclear synergistic mechanism in the prior art, and provides an efficient, safe and green technical scheme for relieving heat stress damage and improving egg quality.

Description

Technical Field

[0001] This invention belongs to the field of feed additive technology, and particularly relates to a feed additive composition containing kaempferol and curcumin and its application in alleviating heat stress in laying hens. Background Technology

[0002] Heat stress is a common environmental stressor in intensive poultry farming. High temperatures can lead to a series of physiological dysfunctions in laying hens, such as decreased feed intake, endocrine disorders, and increased oxidative stress and inflammation, resulting in reduced egg production, decreased eggshell strength, and increased yolk lipid peroxidation. For breeding poultry, the harm of heat stress not only manifests at the maternal level but also negatively impacts offspring health through the "mother-hatching egg-embryo" transmission pathway. Therefore, how to systematically mitigate heat stress damage from the maternal level and ensure the quality of hatching eggs has become a pressing technical challenge for the poultry breeding industry.

[0003] Currently, common methods for alleviating heat stress in laying hens mainly include physical cooling measures and routine nutritional regulation. Physical cooling (such as evaporative cooling pads and misting systems) requires significant investment in equipment and has high operating costs, and its cooling effect is limited by environmental conditions and difficult to maintain stably. Routine nutritional regulation (such as adjusting energy and protein levels and supplementing vitamins and trace elements) mainly focuses on maintaining the basic survival of the hen and the number of eggs laid, lacking a systematic consideration of the intrinsic nutritional quality of hatching eggs and the developmental potential of embryos, and thus failing to meet the needs of poultry production throughout its entire life cycle.

[0004] In recent years, utilizing the antioxidant and anti-inflammatory activities of natural plant polyphenols to alleviate heat stress damage in animals has become a research hotspot. Kaempferol, a flavonoid, has been reported to enhance the endogenous antioxidant defense system by activating the Nrf2 / ARE signaling pathway; curcumin has been reported to reduce the production of pro-inflammatory cytokines by inhibiting the NF-κB inflammatory pathway. However, existing research still has the following significant limitations: First, the dimensions of action are fragmented. Existing technologies mostly focus on kaempferol's enhancement of serum antioxidant enzyme activity or curcumin's reduction of tissue inflammatory factor expression, failing to systematically study and integrate the "oxidative stress relief—inflammation suppression—protection of reproductive endocrine function—ovulation tube eggshell formation—yolk deposition—improvement of hatching egg quality" as a complete physiological cascade. In particular, there is a lack of empirical data to support the connection mechanism between "improvement of maternal redox homeostasis—regulation of oviduct functional gene expression".

[0005] Secondly, there is a lack of research on the synergistic mechanism. Existing reports on the combined use of kaempferol and curcumin are mostly limited to cell experiments or non-heat-stress agonist models. Whether the two have a synergistic effect under continuous heat stress in laying hens, and what their integrated regulatory mechanism is, are all lacking systematic explanation. Most polyphenol compound studies only reach the superficial description that "the combined effect is better than the single component," without elucidating the deep molecular interaction mechanism behind the synergistic effect—whether it is a simple additive effect or a true physiological cascade amplification effect produced by regulating common upstream nodes (such as PI3K / Akt, MAPK, etc.) remains unclear.

[0006] Third, the research models are severely out of touch with actual production practices. Heat stress in breeding poultry is characterized by its chronic, periodic nature and high reproductive load, and its damage exhibits cumulative effects and intergenerational transmission. However, existing technologies mostly employ acute short-term heat exposure models, the intensity and duration of which do not match the actual environment of large-scale farms. This results in insufficient extrapolation and specificity of research conclusions, failing to effectively guide the whole-cycle nutritional regulation strategy centered on "hatching eggs to embryos".

[0007] Furthermore, existing technologies lack research reports on the expression regulation of key functional genes in the oviduct tissue of laying hens under heat stress (such as eggshell formation-related genes CA2 and OCX-32, and the egg white protein gene OVAL), and a systematic evaluation system encompassing "maternal physiological homeostasis—target organ function—final egg quality" has not been established. Existing literature also does not disclose any technical solutions regarding whether kaempferol and curcumin can synergistically improve maternal serum corticosterone and estradiol levels, thereby promoting rapid recovery of production performance after stress.

[0008] In summary, existing technologies for alleviating heat stress damage in laying hens and improving hatching egg quality have prominent problems such as single target, insufficient system integration, unclear synergistic mechanisms, and disconnect between models and production. There is an urgent need to develop a nutritional combination program that can systematically alleviate heat stress damage and improve hatching egg quality from the source of the mother. Summary of the Invention

[0009] To address the aforementioned technical problems, this invention proposes a feed additive composition containing kaempferol and curcumin and its application in alleviating heat stress in laying hens.

[0010] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a feed additive composition for alleviating heat stress in laying hens, wherein the active ingredients of the composition consist of kaempferol (KA) and curcumin (CUR).

[0011] Furthermore, the mass ratio of kaempferol to curcumin is 1:2.

[0012] The present invention also provides the application of the above-mentioned feed additive composition in the preparation of feed to alleviate heat stress in laying hens.

[0013] Furthermore, the feed contains 200 mg / kg of kaempferol and 400 mg / kg of curcumin.

[0014] The feed additive composition of this invention (the active ingredients are kaempferol and curcumin in a 1:2 mass ratio) is not a simple intervention in a single pathway, but rather a systematic alleviation of heat stress damage in laying hens through a multi-level integrated mechanism. Specifically, at the molecular level, kaempferol, as an Nrf2 activator, initiates the Keap1 / Nrf2 / ARE signaling pathway, upregulates the expression of downstream antioxidant enzyme genes such as HO-1 and GSH-Px, and enhances endogenous antioxidant defense; curcumin, on the other hand, inhibits NF-κB p65 nuclear translocation by blocking IκB kinase activity, thereby reducing the production of pro-inflammatory cytokines. The two form a bidirectional positive feedback amplification through the crosstalk mechanism between Nrf2 and NF-κB. Nrf2 upregulates p62 to promote KEAP1 degradation to inhibit NF-κB, while the inhibition of NF-κB reduces the oxidative consumption of Nrf2, thus synergistically achieving an antioxidant and anti-inflammatory effect of "strong scavenging and eliminating the root cause," while downregulating HSP70 to reduce the toxicity of heat stress proteins. At the systemic level, both synergistically regulate the hypothalamic-pituitary-adrenal (HPA) and hypothalamic-pituitary-gonadal (HPG) axes, significantly reducing serum corticosterone levels, relieving the inhibitory effect of high corticosterone on reproductive function, while simultaneously increasing estradiol levels and optimizing energy metabolism. Kaempferol activates the AMPK pathway to improve energy distribution, while curcumin promotes intestinal nutrient absorption, maintaining feed intake and metabolic stability under heat stress, providing sufficient material basis for egg production. At the target organ level, the synergistic effect is precisely delivered to the oviduct: upregulating the expression of carbonic anhydrase 2 (CA2) and eggshell matrix protein OCX-32 genes in the uterus, and enhancing HCO3- in the shell gland. - This invention improves eggshell strength and overall egg quality by enhancing the supply and mineralization capacity of the egg and upregulating the ovalbumin gene (OVAL) in the enlarged part of the egg, thereby promoting egg white protein synthesis. More significantly, improved maternal antioxidant status leads to a significant decrease in malondialdehyde (MDA) content and an increase in total antioxidant capacity in the yolk. By optimizing the yolk microenvironment to program the expression of the Nrf2 gene in the embryonic liver, offspring are endowed with stronger antioxidant defense capabilities, achieving intergenerational health protection from mother to offspring. In summary, this invention systematically addresses the multi-level damage caused by heat stress through an integrated mechanism that integrates molecular network synergy, system homeostasis reconstruction, target organ function enhancement, and intergenerational quality transfer.

[0015] Compared with the prior art, the present invention has the following advantages and technical effects: (1) Significant synergistic effect. This invention is the first to apply kaempferol and curcumin in a 1:2 mass ratio to heat-stressed laying hens. The two showed significant synergistic effects in terms of antioxidation, anti-inflammation, endocrine regulation and egg quality improvement. Two-way ANOVA confirmed that kaempferol and curcumin had significant interaction effects on multiple indicators such as laying rate, eggshell strength, serum total antioxidant capacity, corticosterone level and oviduct key gene expression (P<0.05). The combined use of kaempferol and curcumin was superior to each single addition group in all indicators, and the increase in some indicators (such as serum T-AOC and GSH-Px) exceeded the sum of the increases when the two were added alone, achieving a synergistic gain effect of "1+1>2".

[0016] (2) The feed additive composition of the present invention breaks through the fragmented limitations of existing technologies that only focus on single antioxidant or anti-inflammatory indicators. From the perspective of systems physiology, it completely connects the entire chain of regulatory pathways: oxidative stress and inflammation relief → protection of hypothalamic-pituitary-gonadal axis function → enhanced secretory function of oviduct shell glands → optimization of yolk material deposition → improvement of hatching egg quality. Specifically, the combined use of kaempferol and curcumin can significantly reduce serum corticosterone levels and increase estradiol levels, while upregulating the expression of key genes CA2 and OCX-32 in the uterine region of the oviduct and the OVAL gene in the enlarged region, thereby systematically improving eggshell strength and reducing malondialdehyde content in the yolk. No complete technical solution covering endocrine regulation, oviduct functional gene activation and terminal egg quality improvement has been found in the prior art.

[0017] (3) The feed additive composition of the present invention can not only maintain high production performance during the heat stress period, but also significantly accelerate the recovery speed of various indicators during the normal temperature recovery period. Existing technologies focus more on protection during the stress period and lack a systematic evaluation of the promoting effect during the recovery period.

[0018] (4) The active ingredients kaempferol and curcumin used in this invention are polyphenols derived from natural plants. They have advantages such as high safety, no residue, and no drug resistance. They can be used as a green feed additive in poultry production to replace or reduce the use of chemically synthesized antioxidants and antibiotics, which meets the industrial needs of green and sustainable development in modern animal husbandry.

[0019] In summary, the feed additive composition obtained by the synergistic combination of kaempferol and curcumin in this invention systematically solves the prominent technical problems in the prior art, such as single target and unclear synergistic mechanism. It provides an efficient, safe and industrially scalable green nutrition technology solution for alleviating heat stress damage in breeding poultry and improving the quality of hatching eggs. Attached Figure Description

[0020] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings: Figure 1 The results of the effects of adding KA and CUR individually and in combination on the laying performance of laying hens during the heat stress period and recovery period in the examples and comparative examples are shown. A is the laying rate, B is the average daily egg weight, and C is the feed conversion ratio. Figure 2 The results of the test on the effects of KA and CUR on the dynamic changes of key physiological indicators in the serum of heat-stressed laying hens (A is serum malondialdehyde (MDA) content, B is serum total antioxidant capacity (T-AOC), C is serum corticosterone (CORT) concentration, and D is serum estradiol (E2) concentration). Figure 3 This example compares the expression of key genes in liver and fallopian tube tissues in different groups for both the implementation and comparative examples. Detailed Implementation

[0021] 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.

[0022] 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. Every smaller range between any stated value or intermediate value within a stated range, and 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.

[0023] 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.

[0024] 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.

[0025] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0026] All raw materials used in the embodiments of this invention were commercially available. Kaempferol (standard, purity ≥98%, batch number: KA20250115) was purchased from Shanghai Yuanye Biotechnology Co., Ltd.; curcumin (extract, curcumin content ≥95%, batch number: CUR20241222) was purchased from Xi'an Hongsheng Biotechnology Co., Ltd. Common biochemical reagents used included sodium hydroxide, hydrochloric acid, anhydrous ethanol, methanol, acetone, ethyl acetate, concentrated sulfuric acid, potassium dihydrogen phosphate, trichloroacetic acid, thiobarbituric acid (TBA), and Folin-Ciocalteu reagent. All reagents were of analytical grade and purchased from Sinopharm Chemical Reagent Co., Ltd. Reagent kits supplied by Nanjing Jiancheng Bioengineering Institute were used to test serum and egg yolk oxidative stability indicators, total antioxidant capacity (T-AOC, catalog number A015), malondialdehyde (MDA, catalog number A003), and glutathione peroxidase (GSH-Px, catalog number A005), determining their oxidative defense effects. The following reagents were used in the molecular biology experiments: Total RNA extraction was performed using Trizol reagent (Invitrogen, USA); cDNA synthesis was achieved using the RevertAid First StrandcDNA Synthesis Kit (ThermoScientific, USA); and real-time quantitative PCR was performed using PowerUp™ SYBR™ Green Master Mix (Applied Biosystems, USA). Tissue sample processing reagents: Tissue fixation was performed using 4% paraformaldehyde (Soluble Biotech, Beijing); RNA sample preservation was achieved using RNAwait tissue RNA preservation solution (Soluble Biotech, Beijing).

[0027] This invention was conducted at the Meijin Egg-Laying Chicken Breeding Base in Jiaocheng County, Lüliang City, Shanxi Province, using 320 healthy, stable 32-week-old Hy-Line Brown breeder hens. This age is within the peak egg-laying plateau, with stable egg quality and embryonic developmental potential, making it suitable for evaluating the long-term effects of exogenous additives on egg health. One-way ANOVA confirmed that there were no significant differences in initial body weight and egg production rate among the groups before randomization (P>0.05).

[0028] The technical solution of the present invention will be further illustrated by the following embodiments.

[0029] Example 1 Add KA and CUR to the basal feed at an addition rate of 200 mg / kg KA + 400 mg / kg CUR to obtain the feed, which is denoted as the KA+CUR group.

[0030] Comparative Example 1 Add CUR to the basal feed at a dosage of 400 mg / kg to obtain the feed, which is denoted as the CUR group.

[0031] Comparative Example 2 Add KA to the basal feed at a dosage of 200 mg / kg to obtain the feed, which is denoted as the KA group.

[0032] Comparative Example 3 Basic feed, denoted as CON.

[0033] The group designs in the embodiments and comparative examples of this invention are shown in Table 1. The basic diet is corn-soybean meal, designed according to the "Chicken Feeding Standard" (NY / T33-2004). The basic feed formulas in the embodiments and comparative examples of this invention are shown in Table 2.

[0034] Table 1 Design of each group Table 2 Basic Feed Formulation Note: The premixes in Table 2 contain vitamins, trace elements and other functional additives provided by New Hope Liuhe Co., Ltd.

[0035] Hy-Line Brown laying hens were fed the feeds used in Example 1 and Comparative Examples 1-3. Each group had 8 replicates (pens), with 10 hens per pen, for a total of 80 hens per group. Feeding was managed according to the base's standard procedures, including free access to feed and water, programmed lighting (16L:8D), and ventilation. The experiment lasted 10 weeks, including a 2-week pre-feeding period and an 8-week formal trial period. During the formal trial period, from 9:00 AM to 5:00 PM daily for weeks 1-6, periodic heat stress (33±1℃, 65%±5% humidity) was applied, with normal temperature (25±2℃) maintained for the remaining time. Weeks 7-8 were the recovery period, with normal temperature (25±2℃, 60%±5% humidity) maintained throughout. Feed intake, egg production, and egg weight were recorded daily during the experiment. Sample collection was conducted according to the following protocol: Serum samples: At the end of week 4 (mid-heat stress), week 6 (end of heat stress), and week 8 (end of recovery period), one chicken was randomly selected from each column to collect blood from the wing vein in an empty stomach. The serum was separated and used for subsequent biochemical and hormone level testing. Egg quality and yolk samples: At the same time points (weeks 4, 6, and 8), qualified eggs with normal appearance and no cracks were collected from each group. Two eggs were randomly selected from each column for egg quality testing. Immediately after the test, the yolks were separated and homogenized for analysis of oxidative stability indicators such as yolk lipid peroxidation (MDA) and antioxidant capacity (T-AOC, GSH-Px).

[0036] Tissue Samples: After the formal experiment ended (i.e., at the end of week 8), one chicken was randomly selected from each pen, anesthetized, and slaughtered. Tissue samples of the intact right lobe of the liver and the intact oviduct (focusing on the enlarged portion and uterine portion) were aseptically collected. Some tissues were immediately dissolved in RNA preservation reagent and frozen to -80°C for subsequent RNA extraction and gene expression; the remaining tissues were fixed with 4% paraformaldehyde as reserve samples for morphological studies.

[0037] Performance testing methods: (1) Measurement of production performance indicators During the rearing period, the health status and behavior of each group of laying hens were observed and recorded daily. Using replicates (pens) as units, feed intake, total egg production, egg weight, number of broken and soft-shelled eggs were recorded daily at set times, and hen mortality and culling were also recorded. Data were statistically summarized weekly at the three key stages of the heat stress experiment—weekend 4 (mid-term), weekend 6 (late-term), and weekend 8 (recovery period). The changes in production indicators such as laying rate (%), average daily egg weight (g), average daily feed intake (g), and feed conversion ratio (FCR) were assessed and calculated. The calculation formulas are as follows: (2) Determination of egg quality and yolk oxidative stability At the end of the fourth week of the formal experiment (mid-heat stress), the end of the sixth week of the formal experiment, and the end of the recovery period (mid-heat stress), two qualified eggs with intact appearance and no cracks were randomly selected from eight replicates in each group. A total of 16 eggs / group / time point were used to test the eggshell strength (kg / cm²) using an EQA-500 fully automated egg quality tester (TSS, UK). 2 Eggshell thickness (mm) and egg shape index (major diameter / minor diameter) were measured, and all measurements were performed within 24 hours after sampling.

[0038] After completing the egg quality determination, the egg yolks were separated, and 1.0 g was accurately weighed and added to 9.0 mL of pre-cooled physiological saline. A 10% homogenate was prepared using a tissue homogenizer (10000 r / min, 3×30 s intermittent homogenization) in an ice bath. After centrifugation at 4℃ and 3000×g for 15 min, the supernatant was collected for testing the oxidative stability of the egg yolks. Malondialdehyde (MDA) content was determined using the thiobarbituric acid method (TBA method, kit A003); total antioxidant capacity (T-AOC) was determined using the FRAP (Ferric Reducing Antioxidant Power) method (kit A015); and glutathione peroxidase (GSH-Px) activity was determined using a colorimetric method (kit A005). The Coomassie Brilliant Blue method was used to determine the protein concentration in the homogenate supernatant. MDA and activity were ultimately expressed as the content or activity units corresponding to milligrams of protein (mg prot). All measurements were performed within 4 hours after sample preparation, with two technical replicates for each sample. A standard curve and blank control plot were included for each batch. For egg shape index determination, the major and minor axes of each egg were measured three times, and the average value was taken. The ambient temperature was controlled at 22±1℃, and the relative humidity at 55%±5%.

[0039] (3) Serum biochemical and hormone index determination One chicken was randomly selected from each pen at the mid-heat stress period (end of week 4 of the formal experiment), the end of the heat stress period (end of week 6), and the end of the normal temperature recovery period (end of week 8). Blood was collected from the wing vein in the early morning while the chicken was fasting. After the blood samples were allowed to stand at room temperature for 30 minutes, they were centrifuged at 3000×g for 15 minutes at 4°C. The supernatant serum was carefully separated, aliquoted into 1.5mL centrifuge tubes, and immediately stored at -80°C for analysis.

[0040] Serum antioxidant indicators were determined using kits from Nanjing Jiancheng Bioengineering Institute, strictly following the instructions. Total malondialdehyde (MDA) content was measured using the thiobarbituric acid method (TBA method, kit number: A003), in nanomoles per milliliter of serum (nmol / mL); total antioxidant capacity (T-AOC) was measured using the ferric reduction / antioxidant capacity method (FRAP method, kit number: A015), in millimoles per milliliter of serum (mmol / L), and other indicators. Glutathione peroxidase (GSH-Px) activity was determined by colorimetric method (kit number: A005), and the results are expressed as enzyme activity units per milliliter of serum (U / mL).

[0041] The levels of reproductive hormones in the blood were measured using enzyme-linked immunosorbent assay (ELISA). Corticosterone (CORT) and estradiol (E2) concentrations were analyzed using the dedicated ELISA kits for corticosterone and estradiol from Nanjing Jiancheng Bioengineering Institute (CORT kit number H090, E2 kit number H102). Detection was performed according to existing methods (see Podraza J, Gutowska K, Lenartowicz A, et al. The Role of microRNA in the Regulation of CORTisol Metabolism in the Adipose Tissue in the Course of Obesity [J]. International Journal of Molecular Sciences, 2024, 25(10): 1-13.). During the assay, the procedures in the instructions for the double-antibody sandwich assay kit were strictly followed, including gradient dilution of the standards, sample loading, incubation, washing, color development, and termination of the reaction. Finally, the absorbance of each well was read at a wavelength of 450 nm using a SpectraMaxi3x microplate reader. The sample concentration was then calculated according to the standard curve. The final results were presented in nanograms per milliliter (ng / mL) and picograms per milliliter (pg / mL).

[0042] Each assay included a blank control and a standard control, and every two serum samples were replicated twice. The assay was performed at room temperature (25±2℃), with strict requirements for reaction time and light control to ensure the accuracy and repeatability of the results.

[0043] (4) Tissue sample collection and processing After the formal experiment ended (weekend of week 8), one chicken was randomly selected from each pen (repeated). After fasting for 12 hours (with free access to water), the chicken was euthanized by exsanguination from the neck, and necropsy was performed quickly. Liver tissue: A tissue block of approximately 1.0 cm³ from the middle of the right lobe of the liver was aseptically collected. After equalizing the sample, one 500 μL portion was immediately placed in a cryogenic RNA preservation reagent, briefly refrigerated, and then placed in a -80°C cryogenic storage device for further total RNA extraction and gene expression studies. The other 500 μL portion was placed in 4% paraformaldehyde phosphate buffer (pH 7.4) and fixed at 4°C for 24–48 hours, which can be used for paraffin embedding and alternative histological detection.

[0044] Oviduct tissue: The oviduct was completely isolated, with a focus on collecting tissue from the enlarged portion (the main area secreting egg white protein) and the uterine portion (where the shell glands are located, responsible for eggshell formation). Approximately 1.0 cm samples were taken from each region and gently washed with pre-cooled physiological saline. Each sample was divided into two portions for subsequent experiments. One portion was immediately immersed in RNA protection reagent, briefly thawed on ice, and then transferred to a -80°C cryopreservation facility for RNA extraction. The other portion was immobilized in a 4% paraformaldehyde solution for subsequent histological studies.

[0045] All cryopreserved samples used for molecular biology assays were rapidly frozen in liquid nitrogen for 5-10 minutes before long-term storage at -80°C to maximize the preservation of RNA integrity. Tissues used for fixation were dehydrated, cleared, and paraffin-embedded according to standard procedures after fixation to prepare tissue blocks for sectioning.

[0046] (5) Detection of gene expression levels To elucidate the molecular mechanisms of kaempferol and curcumin, quantitative real-time PCR was used to evaluate the transcriptional levels of the target genes in the liver and fallopian tubes. RNA extraction and quality control were performed: approximately 50 mg of liver and fallopian tube tissue samples stored at -80℃ were rapidly ground into powder in liquid nitrogen. Total RNA was extracted from the tissues using Trizol reagent. The concentration and purity of the extracted RNA samples were accurately detected using ultra-micro spectrophotometry, with the A260 / A280 ratio strictly limited to between 1.8 and 2.0. A acceptable ratio range indicates that the extracted RNA has met the purity standards for experimental research. Simultaneously, agarose gel electrophoresis was used to detect RNA integrity; clear 28S and 18S rRNA bands were observed, indicating good RNA integrity.

[0047] cDNA synthesis: Take 1 μg of qualified total RNA and synthesize cDNA using a reverse transcription kit. The reaction volume is 20 μL, and the procedure must be strictly followed according to the manufacturer's instructions. The synthesized cDNA is stored at -20℃ for subsequent PCR amplification.

[0048] Real-time quantitative PCR: PCR reactions were performed on a real-time quantitative PCR instrument. Detection was performed using the SyBr Green fluorescent dye method. The reaction volume was 20 μL, which included premixed SyBr GreenMasterP, custom-made upstream and downstream primers, cDNA sample, and nuclease-free water. Primer sequences are shown in Table 3. All primers were custom-synthesized by Sangon Biotech (Shanghai) Co., Ltd., ensuring the purity and reliability of the synthesis.

[0049] The experimental procedure was as follows: First, a pre-denaturation treatment was performed at 95℃ for 2 minutes, followed by 40 cycles of PCR amplification. Each cycle included three steps: 15S DNA denaturation at 95℃; primer annealing and DNA extension at 60℃ for 1 minute to complete primer binding to the template and synthesis of a new strand; and product specificity was detected by melting curve analysis. For quantitative analysis of the target gene expression level, β-Actin was selected as an internal reference gene, and relative expression levels were calculated using 2^-ΔΔCt. The experimental results were measured three times for each sample.

[0050] For RT-qPCR, each sample was performed in triplicate, with the standard deviation of Ct values ​​controlled below 0.3. Melting curves showed a single peak, indicating no nonspecific amplification. Relative expression levels were calculated using the 2^-ΔΔCt method, with the CON group serving as the calibration sample.

[0051] Table 3 Primer sequences for real-time quantitative PCR Statistical analysis: Experimental data were statistically analyzed using SPSS 26.0 software. Two-way ANOVA was used to systematically study the main effects and interaction effects of KA and CUR on various production performance indicators, egg quality traits, yolk oxidative stability evaluation, serum biochemical parameters, and tissue gene expression profiles. When the interaction effect was significant, Duncan's method was used for multiple comparisons to distinguish specific differences. When the interaction effect was not significant but the main effect was significant, a summary analysis of the treatment levels of KA and CUR was performed to accurately elucidate the independent effects of KA and CUR on each indicator. To further explore the intrinsic relationships among the indicators, Pearson correlation analysis was performed on key production performance, egg quality, serum biochemical parameters, and gene expression indicators. Graphs and charts were generated using Graph PadPrism 8.02 software. P < 0.05 was used as the criterion for statistical significance. All results are expressed as mean ± standard deviation (x̄ ± SD). All data underwent normality tests (Shapiro-Wilk test) and homogeneity of variance tests (Levene's test) before analysis. Welch correction was used for heterogeneous variances.

[0052] Performance test results: (1) Effects of KA and CUR on the production performance of heat-stressed breeding hens The results of the tests on the effects of KA and CUR on the production performance of heat-stressed laying hens in the examples and comparative examples are shown in Table 4. It can be seen that both components, when added individually, can partially alleviate the negative impact of heat stress on laying efficiency, but when added together, they have a more significant synergistic promoting effect. At the end of the heat stress period, in week 6, the laying rate and average egg weight of the KA+CUR group were significantly higher than those of the control group and each of the individual addition groups (P<0.05), and there was a significant interaction between KA and CUR in these two indicators (P<0.05). There was no significant difference in feed intake among the groups, indicating that the performance improvement mainly stemmed from metabolic improvements.

[0053] At the end of the recovery period in week 8, the egg production rate and egg weight of the KA+CUR group recovered faster, and the feed conversion ratio was significantly lower than that of the control group (P<0.05), indicating that the KA+CUR group has a synergistic advantage in improving feed conversion efficiency and promoting recovery.

[0054] In summary, the combined addition of KA and CUR can synergistically mitigate the adverse effects of heat stress on the laying hen's production performance, while also enhancing the laying hens' ability to recover after stress.

[0055] Table 4. Results of test on the effects of KA and CUR on the production performance of heat-stressed breeding hens. Data in Table 4 are expressed as mean ± standard deviation. Different superscript letters for the same indicator at the same time point indicate significant differences (P < 0.05). The P-value represents the significance of the interaction between KA and CUR. The same applies to the following tables.

[0056] Figure 1The figures show the effects of kaempferol (KA) and curcumin (CUR) alone and in combination on the laying performance of hens during the heat stress and recovery periods in the examples and comparative examples. A represents the laying rate, B represents the average daily egg weight, and C represents the feed conversion ratio (heat stress treatment period was weeks 1-6 of the formal experiment, and weeks 7-8 were the recovery period. Time points 4, 6, and 8 in the figure correspond to the middle of heat stress (end of week 4), the end of heat stress (end of week 6), and the end of the recovery period (end of week 8), respectively. Different lowercase letters at the same time point indicate significant differences between treatment groups (P<0.05). CON: control group; KA: kaempferol group; CUR: curcumin group; KA+CUR: combined addition group, the same below). It can be seen that chronic heat stress severely impairs the laying performance of breeder hens, and the addition of kaempferol and curcumin to the diet, especially when used together, can significantly regulate and alleviate this condition. Throughout the entire trial, the combined supplementation group (KA+CUR) consistently demonstrated the best performance in key production indicators. Specifically, at the peak of heat stress (week 6), the KA+CUR group exhibited the highest egg production rate and egg weight, the lowest feed conversion ratio, and the best feed conversion efficiency. During the recovery period at week 8, this group showed the fastest and highest recovery in production indicators, indicating that both supplementation and CUR worked together to promote functional recovery after stress.

[0057] (2) Effects of KA and CUR on hatching egg quality and yolk oxidative stability Table 5 shows the effects of KA and CUR on egg quality during the heat stress and recovery periods in the examples and comparative examples. Table 6 shows the effects of KA and CUR on yolk oxidative stability in the examples and comparative examples. As can be seen from Tables 5 and 6, adding KA and CUR individually or in combination significantly improves eggshell strength, thickness, and yolk oxidative state. The KA+CUR group showed the best performance in all relevant indicators, confirming their synergistic effect. During periods of high heat stress, the eggshell strength of the KA+CUR group was 3.88 kg / cm². 2The levels of malondialdehyde (MDA) in the egg yolks of this group were significantly higher than those of the control group (CON) and each single-addition group, and the decrease in MDA with increasing stress intensity was the smallest. Simultaneously, this group had the lowest MDA content in the egg yolks, and the reduction in MDA was significantly greater than in the single-treatment groups, indicating a significant synergistic effect between the two treatments in inhibiting lipid peroxidation. Furthermore, the antioxidant capacity (T-AOC) and glutathione peroxidase (GSH-Px) of the KA+CUR group reached the highest levels, indicating that the combined treatment of KA and CUR can effectively inhibit the oxidative damage of egg yolks caused by heat stress, protect the integrity of the eggshell, and significantly improve the internal quality, external characteristics, and oxidative stability of hatching eggs treated with the combined KA and CUR treatment.

[0058] Table 5. Effects of KA and CUR on egg quality during the heat stress and recovery periods in the examples and comparative examples. Table 6. Effects of KA and CUR on the oxidative stability of egg yolk in the Examples and Comparative Examples (3) Effects of KA and CUR on serum biochemical indicators Table 7 shows the results of the determination of the effects of KA and CUR on serum biochemical indicators in each group of the examples and comparative examples.

[0059] Table 7. Results of the determination of the effects of KA and CUR on serum biochemical indicators in each group of the examples and comparative examples. Table 7 shows that at the mid-stage of heat stress (week 4, 36 weeks of age), compared with the CON group, the KA group, CUR group, and KA+CUR group significantly reduced serum MDA levels by 10.8%, 6.3%, and 16.6%, respectively (P<0.05), and also significantly increased the activities of T-AOC and GSH-Px. The KA+CUR group showed optimal results for all indicators, and the increase in T-AOC and GSH-Px was significantly greater than the sum of the increases when added individually to the KA and CUR groups, indicating a synergistic amplification effect.

[0060] Regarding stress hormones, CORT levels decreased significantly in all treatment groups, with the KA+CUR group showing the most significant reduction, decreasing by 16.9% compared to the CON group; estradiol (E2) concentrations increased significantly, indicating that all treatment groups could regulate stress hormones. The bidirectional regulatory effect of decreasing CORT and increasing E2 was most pronounced in the combined treatment group, and was significantly stronger than the effect of a single additive.

[0061] At the end of the heat stress period, week 6 (38 weeks of age), the combined group still maintained the best antioxidant state, with MDA content 21.4% lower than that of the CON group, and the highest activities of T-AOC and GSH-Px (P<0.05). At this time, the CORT levels of the KA group and CUR group were 6.0 ng / mL and 6.3 ng / mL, respectively, both 6.9 ng / mL lower than the mid-term, but still showing some recovery, suggesting that the body may have a stress response under continuous heat load; while the CORT of the KA+CUR group was still 5.3 ng / mL, significantly lower than that of the mid-term, and the estradiol (E2) levels of all groups were also decreasing, but the E2 of the KA+CUR group was 111.2 pg / mL, significantly higher than that of the control group, both at 73.0 pg / mL (P<0.05), further verifying the synergistic regulatory effect.

[0062] At week 8 of the recovery period, when the patient was 40 weeks old, the antioxidant index of the KA+CUR group remained in the lead (P < 0.05). The level of corticosterone (CORT) was 4.2 ng / mL, which was significantly lower than that of the CON group (5.3 ng / mL). The concentration of estradiol (E2) was 133.0 pg / mL, which was significantly higher than that of the CON group (96.2 pg / mL). This indicates that the combined addition has superior characteristics in terms of endocrine homeostasis during the recovery period.

[0063] In summary, KA and CUR work together to enhance antioxidant capacity, dynamically regulate CORT and E2 levels, and jointly promote heat stress adaptation and physiological recovery.

[0064] Figure 2 The results of the test on the effects of KA and CUR on the dynamic changes of key physiological indicators in the serum of heat-stressed laying hens (A is serum malondialdehyde (MDA) content, B is serum total antioxidant capacity (T-AOC), C is serum corticosterone (CORT) concentration, and D is serum estradiol (E2) concentration). Figure 2The results showed that KA and CUR synergistically improved the serum biochemical status of heat-stressed laying hens. At all time points, the combined KA+CUR group exhibited the lowest serum MDA content and the highest T-AOC level, demonstrating the strongest antioxidant capacity and minimal oxidative damage. Regarding stress hormones, KA+CUR reduced CORT levels during the mid-heat stress period (week 4), effectively alleviating the acute heat stress response. Towards the end of the heat stress period (week 6), sustained high-temperature loading increased CORT in all groups, while E2 was suppressed, but relatively low CORT and relatively high E2 levels were still maintained. During the recovery period (week 8), the KA+CUR group showed the fastest CORT decline, the best E2 recovery, and better restoration of reproductive endocrine homeostasis. In conclusion, KA and CUR effectively mitigated heat stress damage and promoted physiological recovery by synergistically enhancing antioxidant capacity and dynamically regulating stress and reproductive hormone levels. Changes in serum biochemical indicators reflect systemic physiological responses, but the molecular mechanisms of KA and CUR still need to be verified at the tissue level. Therefore, we further investigated the expression levels of key genes in the liver and fallopian tubes.

[0065] (4) Effects of KA and CUR on the expression of key genes in hepatobiliary tissues To investigate the molecular mechanisms by which KA and CUR alleviate heat stress, a comprehensive analysis of core genes in the liver and oviduct tissues was conducted. Table 8 shows the results of the test on the effects of KA and CUR on the expression of key genes in the tissues of laying hens in each group of the examples and comparative examples.

[0066] Table 8. Results of the test on the effects of KA and CUR on the expression of key genes in the tissues of laying hens in each group of the examples and comparative examples. As shown in Table 8, the liver heat stress marker gene HSP70 and the inflammatory pathway gene NF-κBp65 were significantly reduced in the KA+CUR synergistic treatment group (KA+CUR group), while the antioxidant genes nrf2 and HO-1 were significantly increased (P<0.05). This indicates that the KA+CUR group can reduce heat stress damage by regulating the expression of related genes. In the oviduct tissue, the KA+CUR group significantly increased the expression levels of eggshell formation-related genes CA2, *OCX-32*, and the egg white protein gene OVAL. Two-way ANOVA showed a significant interaction between KA and CUR in regulating the expression of most of these genes (P<0.05), confirming a synergistic regulatory effect at the molecular level. This is consistent with the trend of improved production performance and egg quality.

[0067] Figure 3For the comparison of key gene expression in liver and oviduct tissues of different groups in the examples and comparative examples (different lowercase letters on the same column indicate significant differences (P<0.05)), it can be seen that under heat stress (CON group), the expression of most key genes in laying hens is at a relatively low level. The combined addition of KA and CUR showed a significant synergistic regulatory effect: the combined group not only significantly increased the expression of antioxidant genes (Nrf2, HO-1) and eggshell formation genes (CA2, OCX-32), with a particularly significant upregulation of Nrf2, but also effectively inhibited the expression levels of heat stress genes (HSP70) and inflammation genes (NF-κBp65). This indicates that the two combined can jointly regulate gene expression in multiple pathways of "antioxidation-anti-inflammation-eggshell formation," comprehensively alleviating the damage caused by heat stress at the molecular level.

[0068] (5) Correlation analysis among key indicators To systematically explore the synergistic regulatory mechanism of kaempferol (KA) and curcumin (CUR) on improving the production performance, egg quality, and physiological traits of heat-stressed laying hens, Pearson correlation analysis was used, focusing on key productivity indicators, egg quality indicators, blood metabolism indicators, and embryonic gene transcription levels to investigate their intrinsic correlations. Table 9 shows the correlation analysis results among key indicators of heat-stressed laying hens (the data in the table are Pearson correlation coefficients, and *, **, and *** indicate significant correlation at the 0.05, 0.01, and 0.001 levels (two-sided), respectively. n=32).

[0069] Table 9. Correlation analysis results among key indicators of heat stress in laying hens As shown in Table 9, there is a close intrinsic correlation among the key indicators, forming a complete chain from maternal physiological condition to offspring gene expression. The egg production rate and feed conversion ratio showed a very significant negative correlation (r=-0.79, P<0.01), while the egg production rate and eggshell strength showed a positive correlation (r=0.41, P<0.05), indicating that improvements in production performance, feed utilization efficiency, and eggshell quality occurred simultaneously.

[0070] Regarding oxidative stress, yolk MDA content was significantly negatively correlated with egg production rate and eggshell strength (r=-0.52, -0.48, P<0.01), and positively correlated with serum CORT levels (r=0.57, P<0.01). In contrast, serum T-oc was significantly positively correlated with egg production rate and eggshell strength (R=0.58, 0.39, P<0.05), but significantly negatively correlated with yolk oxidative stability (R=-0.66, P<0.001). This indicates that when the maternal antioxidant capacity is improved, it can not only alleviate its own oxidative damage, but also affect the yolk oxidative stability and eggshell strength.

[0071] Stress and reproductive endocrine regulation also showed a regular correlation. Serum CORT was negatively correlated with egg production rate and serum T-AOC (r=-0.51, -0.62, P<0.01), indicating that heat stress-activated HPA axis inhibits antioxidant status and production performance. Serum E2 was positively correlated with egg production rate and serum T-AOC (r=0.46, 0.49, P<0.05), suggesting that a good antioxidant state helps maintain reproductive endocrine homeostasis.

[0072] Of particular note was the significant correlation between Nrf2 gene expression in embryonic liver tissue and several maternal indicators: a positive correlation with egg production rate and serum T-AOC (r=0.43, 0.51, P<0.05), and a negative correlation with yolk MDA and serum CORT (r=-0.48, -0.47, P<0.05). This directly confirms that programming the maternal antioxidant status and stress level through the yolk microenvironment can improve the antioxidant defense capabilities of offspring embryos.

[0073] In summary, the correlation analysis established a complete physiological transmission chain: "enhanced maternal antioxidant capacity (elevated serum T-AOC) → reduced stress response (decreased serum CORT) → reduced yolk oxidative damage (decreased yolk MDA) → improved eggshell quality (increased eggshell strength) → improved production performance (increased egg production rate, decreased feed conversion ratio) → embryonic antioxidant programming (increased embryonic liver Nrf2)." The synergistic effect of KA and CUR is very likely achieved through the synchronous optimization of multiple key nodes in this chain, thereby realizing systemic health regulation from mother to offspring.

[0074] The above are merely preferred embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention.

Claims

1. A feed additive composition for alleviating heat stress in laying hens, characterized in that, The active ingredients of the composition consist of kaempferol and curcumin.

2. The feed additive composition for alleviating heat stress in laying hens according to claim 1, characterized in that, The mass ratio of kaempferol to curcumin is 1:

2.

3. The use of a feed additive composition as described in any one of claims 1 to 2 in the preparation of feed for alleviating heat stress in laying hens.

4. The application of the feed additive composition according to claim 3 in the preparation of feed to alleviate heat stress in laying hens, characterized in that, The feed contains 200 mg / kg of kaempferol and 400 mg / kg of curcumin.

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