Phosphorylated protein marker for fasting-induced chicken blood testis barrier remodeling, screening method and application

Abstract

The invention relates to the technical field of poultry reproductive biology, and particularly discloses a phosphorylated protein marker for fasting-induced chicken blood testis barrier remodeling, a screening method and application. The marker comprises a Ser1573 phosphorylation site which is tightly connected with the protein ZO-1 and a Ser1620 phosphorylation site of the ZO-1. The screening method comprises the following steps: collecting testicular tissues of cocks at key time points of fasting and recovering foraging, carrying out proteomics and phosphorylation detection, analyzing and screening different phosphorylation sites in combination with bioinformatics, and carrying out multi-dimensional verification through Western blot, immunofluorescence and cell function experiments. The invention provides a key molecular marker for evaluating the testis functional state of the cock and optimizing a limited feeding scheme in breeding of the breeder cock, and has important production and application values.

Description

Technical Field

[0001] This invention relates to the field of avian reproductive biology, and in particular to a phosphorylated protein marker, screening method, and application for fasting-induced blood-testis barrier remodeling in chickens. Background Technology

[0002] The blood-testis barrier (BTB) is formed by adjacent Sertoli cells connected by tight junctions (TJs), gap junctions (GJs), desmosomes, and specialized basal cytoplasmic structures (ESs). It divides the seminiferous epithelium into a luminal and basal region. Only leptotene spermatocytes that cross the BTB into the luminal region can undergo meiosis. Its main functions are to maintain the stability of the microenvironment within the seminiferous tubules, protect spermatogenic cells from harmful external substances, and promote spermatogenesis. In rooster breeding, appropriate feed restriction (fasting) is an important measure to improve reproductive performance. Through fasting induction, spermatogenesis in roosters can be reversibly restored. The integrity of the blood-testis barrier is an indispensable guarantee for ensuring spermatogenesis. Therefore, screening for molecular markers that can accurately reflect the fasting-induced blood-testis barrier remodeling process is of great significance for optimizing feed restriction programs for roosters and improving breeding efficiency.

[0003] Currently, research on the blood-testis barrier (BTB) focuses primarily on mammals, with limited studies on birds, especially roosters. Existing techniques mainly assess BBT status through morphological observation or by detecting the expression levels of a few known tight junction proteins (such as ZO-1 and Claudin-11), but these methods struggle to accurately reflect the dynamic processes of barrier remodeling. Proteomics and phosphoproteomics technologies provide powerful tools for systematically screening key molecular markers. By comprehensively analyzing changes in protein expression and phosphorylation modifications, specific markers closely related to physiological processes can be identified. Summary of the Invention

[0004] The purpose of this invention is to provide a phosphorylated protein biomarker, screening method and application for fasting-induced blood-testis barrier remodeling in chickens, providing a scientific basis for accurately assessing the blood-testis barrier status of roosters and optimizing feed restriction programs.

[0005] To achieve the above objectives, on the one hand, the present invention provides a phosphorylated protein biomarker for fasting-induced remodeling of the blood-testis barrier in chickens, the biomarker comprising the Ser1573 phosphorylation site of tight junction protein ZO-1 and / or the Ser1620 phosphorylation site of ZO-1.

[0006] On the other hand, the present invention also provides a method for screening phosphorylated protein markers of fasting-induced blood-testis barrier remodeling in chickens, comprising the following steps: S1. Sample Collection: Healthy adult "Yufen No. 1" H-strain roosters with uniform weight were selected. The experiment was divided into two phases: a pre-trial period and a regular feeding period. The regular feeding period included a fasting period and a recovery feeding period. The fasting period was divided into two stages: fasting and water restriction, and fasting and water restriction treatment. The recovery feeding period was also divided into two stages: first, a gradual transition with quantitative feeding and water, and then a return to free feeding and drinking. Testicular tissue samples were collected from the roosters at key time points during the experiment. S2. Protein extraction and enzymatic hydrolysis: Total protein was extracted from F0, F15, and R36 testicular tissues, and after reduction alkylation treatment, peptide fragments were obtained by trypsin hydrolysis. S3. Enrichment of phosphorylated peptides: Phosphorylated peptides were enriched using TiO2 affinity chromatography. S4. Proteomics and phosphoproteomics analysis: Liquid chromatography-tandem mass spectrometry was used to detect peptides and phosphorylated peptides to obtain protein expression levels and phosphorylation modification data; S5. Differential Analysis: By comparing and analyzing differences in protein expression and phosphorylation modification using bioinformatics software, significantly different phosphorylated proteins and sites are screened out. S6. Functional Validation: Western blot and immunofluorescence techniques were used to verify the expression and localization changes of differentially phosphorylated proteins. Combined with IP assays and cellular and molecular methods, specific biomarkers related to blood-testis barrier remodeling were identified.

[0007] Furthermore, in S1, after resuming feeding, the sperm volume, sperm motility, sperm density, sperm abnormality rate, and effective sperm count were continuously measured to evaluate the remodeling of reproductive performance.

[0008] Furthermore, in S5, bioinformatics analysis includes differentially expressed proteins, differentially phosphorylated proteins, and KEGG pathway enrichment.

[0009] This invention also provides an application of phosphorylated protein markers of fasting-induced blood-testis barrier remodeling in assessing the remodeling status of the blood-testis barrier. The application determines the integrity of the blood-testis barrier by detecting the Ser1573 phosphorylation site, the Ser1620 phosphorylation site, and the overall phosphorylation level of ZO-1 in testicular tissue.

[0010] This invention also provides an application of a phosphorylated protein biomarker for fasting-induced blood-testis barrier remodeling in optimizing the feed restriction program for breeding roosters. By detecting the phosphorylation level of the biomarker, it is helpful to dynamically adjust the fasting duration and refeeding nutrient ratio to maintain the stability of blood-testis barrier function.

[0011] Compared with the prior art, the present invention has the following advantages and technical effects: (1) This invention is the first to systematically screen specific phosphorylated protein markers for fasting-induced blood-testis barrier remodeling in chickens using proteomics and phosphatomics technologies, which makes up for the lack of precise molecular markers in the existing technology and provides a new perspective for a deeper understanding of the regulatory mechanism of the blood-testis barrier in birds.

[0012] (2) The selected biomarker combination (ZO-1 P-Ser1573 and ZO-1 P-Ser1620) can dynamically reflect the remodeling process of the blood-testis barrier and can be used as a molecular indicator to assess the functional status of rooster testes. It can be used for rapid detection in breeding roosters, providing a scientific basis for precise regulation of feed restriction programs, and helping to improve the reproductive performance and economic benefits of breeding roosters. The screening method of this invention combines high-throughput omics technology and multi-dimensional experimental verification to ensure the reliability and practicality of the biomarkers, and can provide a reference for the screening of blood-testis barrier-related biomarkers in other poultry or mammals.

[0013] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0014] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments of the present invention 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.

[0015] Figure 1 The results represent the dynamic changes in semen quality of roosters during the fasting induction process. A represents the changes in sperm motility, B represents the changes in sperm density, C represents the changes in semen volume, D represents the changes in sperm abnormality rate, and E represents the changes in effective semen volume. Figure 2 Immunofluorescence of ZO-1 / WT1 / DAPI at different stages during fasting induction; Figure 3 The number of differentially expressed proteins (F15 vs F0, F15 vs R36, and R36 vs F0) during fasting induction was statistically analyzed. Figure 4 KEGG pathway enrichment analysis of differentially expressed proteins F15 vs F0, F15 vs R36, and R36 vs F0 during fasting induction. A is the KEGG enrichment map of differentially expressed proteins F15 vs F0, B is the KEGG enrichment map of differentially expressed proteins F15 vs R36, and C is the KEGG enrichment map of differentially expressed proteins R36 vs F0. Figure 5The number of differentially phosphorylated proteins in F15 vs F0, F15 vs R36, and R36 vs F0 during fasting induction was statistically analyzed. Figure 6 For the KEGG pathway enrichment analysis of differentially phosphorylated proteins F15vsF0, F15vsR36, and R36vsF0 during fasting induction, A is the KEGG enrichment map of differentially phosphorylated proteins F15vsF0, B is the KEGG enrichment map of differentially phosphorylated proteins F15vsR36, and C is the KEGG enrichment map of differentially phosphorylated proteins R36vsF0. Figure 7 To screen key genes and pathways for fasting-induced blood-testis barrier remodeling using proteomics and protein phosphatomics, A shows Venn diagrams of differentially expressed proteins and differentially phosphorylated proteins among the F0, F15, and R36 groups; B shows Venn diagrams of differentially expressed proteins and differentially phosphorylated proteins (differential molecules) between F15 and F0, and between F15 and R36; and C shows KEGG enrichment analysis of the differential molecules. Figure 8 The abundance and phosphorylation levels of key proteins in the testicular tissue of roosters during fasting induction are shown in Figure 1. A represents the protein abundance changes at different stages during fasting induction, B represents the abundance changes of differentially phosphorylated proteins at different stages during fasting induction, C represents the ratio of differentially phosphorylated protein abundance to the corresponding total protein abundance, D represents the abundance changes of ZO-1 (TJP1) protein during fasting induction, E represents the phosphorylation abundance changes of ZO-1 (TJP1) protein during fasting induction, and F represents the ratio of phosphorylation abundance to protein abundance of ZO-1 (TJP1) protein during fasting induction. Figure 9 The effects of different concentrations of androgens (0%, 1%, 2.5%, 5%, 7.5%, 10%) on ZO-1 expression in primary chicken Sertoli cells were investigated. In Figure A, Western blot (WB) results of ZO-1 protein in each treatment group are shown, and B is a quantitative graph of the gray value of ZO-1 protein bands in Figure A. Figure 10 The effect of different concentrations of androgens on the levels of ZO-1 protein and its serine phosphorylation (P-Ser) in primary chicken Sertoli cells is shown in Figure A, where A represents the Western blot (WB) of ZO-1 and P-Ser IP after treatment with different concentrations of androgens, and B represents the quantitative WB of ZO-1 and P-Ser IP after treatment with different concentrations of androgens. Figure 11The effects of different concentrations of androgens on the expression of ZO-1 protein and the level of ZO-1 phosphorylation (P-ZO-1) in chicken primary supporting cells at specific times (1h, 12h, 24h) were investigated. In the figure, A is the WB detection result of each treatment group, B is the quantitative graph of the gray value of the ZO-1 protein band in figure A, and C is the quantitative graph of the gray value of the P-ZO-1 band in figure A. Detailed Implementation

[0016] The technical solution of the present invention will be further described below through embodiments.

[0017] Unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0018] Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of this invention. Experimental methods in the following embodiments that do not specify specific conditions are generally determined according to national standards. Experimental instruments, equipment, and reagents in the following embodiments that do not specify their sources are all commercially available materials.

[0019] Unless otherwise defined or stated, all technical and scientific terms used in this invention have the same meaning as those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein can be applied to the methods of this invention. It should be noted that, unless otherwise specified, the embodiments and features described in this invention can be combined with each other.

[0020] The “Yufen No. 1” H-strain rooster used in this invention experiment was sourced from the Poultry Germplasm Resource Farm of Henan Agricultural University.

[0021] Example 1 1.1 Experimental Design and Sample Collection: Thirty-five healthy, uniformly weighted adult "Yufen No. 1" H-strain roosters aged 500 days were selected. The experiment lasted 69 days, divided into a 7-day pre-trial period and a 62-day regular feeding period (15 days of fasting + 47 days of resumed feeding). The fasting period was divided into two phases: the first phase was 3 days of fasting + 3 days of water deprivation, with 8 hours of light per day; the second phase was 12 days of fasting + 12 days of water deprivation, with 8 hours of light per day. The resumption of feeding was divided into two phases: the first phase was 15g of feed per day + water deprivation, with 8 hours of light + 0.5 hours of light per day; the second phase continued until feed intake reached 120g, with 16 hours of light per day, after which free access to feed and water was provided. At the critical time points F0 (the day before fasting and water deprivation), F15 (the 15th day of fasting), and R36 (the 36th day of resumed feeding), two roosters were randomly selected, euthanized, and their testicular tissue was collected and quickly frozen in liquid nitrogen.

[0022] 1.2 Protein extraction and enzymatic hydrolysis: Take 0.1g of testicular tissue, add RIPA lysis buffer (containing phosphatase inhibitor), grind in an ice bath, centrifuge at 4℃ (12000g, 15 minutes), collect the supernatant, and determine the protein concentration using the BCA method. Take 50μg of total protein, add DTT to a final concentration of 10mM, reduce at 56℃ for 30 minutes; add iodoacetamide to a final concentration of 20mM, alkylate in the dark for 30 minutes; digest with trypsin at an enzyme:protein ratio of 1:50 overnight at 37℃.

[0023] 1.3 Enrichment of phosphorylated peptides: Phosphorylated peptides were enriched using TiO2 microsphere affinity chromatography. The enzymatically digested peptides were dissolved in a loading buffer (80% acetonitrile, 6% trifluoroacetic acid), mixed with TiO2 microspheres, and incubated for 30 minutes. The microspheres were collected by centrifugation and washed sequentially with Washing buffer I (80% acetonitrile, 6% trifluoroacetic acid, 50 mM oxalic acid) and Washing buffer II (80% acetonitrile, 0.1% trifluoroacetic acid). Finally, the phosphorylated peptides were eluted with Elution buffer (5% ammonia), and then vacuum-dried for later use.

[0024] 1.4 LC-MS / MS Analysis: Unphosphorylated peptides and enriched phosphorylated peptides were dissolved separately in 0.1% formic acid solution and separated using a nano-liquid chromatography system (Easy-nLC 1200) with a C18 reversed-phase column (75 μm × 25 cm). Mobile phase A was 0.1% formic acid aqueous solution, and mobile phase B was 0.1% formic acid acetonitrile solution. Gradient elution was used (0–5 min, 5% B; 5–80 min, 5%–35% B; 80–85 min, 35%–90% B; 85–90 min, 90% B) at a flow rate of 300 nL / min. The peptides were then electrospray ionized and detected by a QE-HF mass spectrometer with a scan range of 350–1600 m / z. The primary mass spectrometry resolution was 60,000, and the secondary mass spectrometry was performed in data-dependent mode with a resolution of 15,000 and a collision energy of 30 eV.

[0025] 1.5 Difference Analysis: Raw mass spectrometry data were retrieved using MaxQuant software (v1.6.10.43) and searched for databases (UniProt Gallusgallus database). Variable modifications were set to phosphorylation (Ser / Thr / Tyr) and oxidation (Met), while the fixed modification was carbamidomethyl (Cys), with a false detection rate (FDR) <1%. Differential analysis was performed using Perseus software to screen for differentially expressed proteins with a fold change >2 and p < 0.05 between the control and fasting treatment groups, as well as differentially phosphorylation sites with a fold change >2 and p < 0.05. To investigate the reasons for semen quality remodeling, differentially expressed molecules were screened based on a fold change of F15 vs F0 and F15 vs R36 protein phosphorylation abundance / protein abundance FC > 2. GO functional annotation and KEGG pathway enrichment analysis were performed using the DAVID database, focusing on pathways involved in tight junctions.

[0026] 1.6 Functional Verification: (1) Western blot verification: ① Take testes from 3-5 week old chickens, remove the membrane and blood vessels, wash 3 times with PBS containing 2% penicillin and antibiotics, and cut into pieces. Small pieces were digested with collagenase IV (1 mg / L) at 37°C for 40-50 min, followed by digestion with 0.25% trypsin-EDTA for 6-8 min, filtered through a 300-mesh sieve, and cultured in DMEM / F12 medium containing 10% FBS. After 24 h, the medium was hypotonic (PBS: distilled water = 1:2, 3 min), and then replaced with fresh medium for continued culture. ② Based on DMEM / F12 medium, cell culture media with different serum concentrations were prepared. 500 μL of penicillin and streptomycin was added to a 50 mL centrifuge tube, followed by 0 μL (0%), 500 μL (1%), 1.25 mL (2.5%), 2.5 mL (5%), 3.75 mL (7.5%), and 5 mL (10%) of serum. The medium was then replenished to 50 mL. When the supporting cells reached 80% confluence in the 6-well plate, the cells were treated with different concentrations of serum for 1 h, 3 h, 6 h, 12 h, 24 h, and 48 h. Cells were then collected to detect ZO-1 protein expression and its IP with p-Ser.

[0027] (2) Immunofluorescence verification: F0, F15 and R36 testicular tissues were prepared into frozen sections. After the frozen sections were permeabilized, they were blocked with 5% BSA, and ZO-1 and WT1 (supporting cell markers) were added and incubated at 4℃. Secondary antibody was incubated at room temperature in the dark, and the nuclei were stained with DAPI. The signal was collected under a microscope.

[0028] Experimental results: Monitoring the semen quality of breeding roosters during the fasting induction process revealed that the sperm motility of F15 breeding roosters ( Figure 1(A) Sperm density ( Figure 1 (B) Semen volume ( Figure 1 C), effective sperm count ( Figure 1 Both D and D levels decreased significantly; after feeding resumed, these indicators recovered to varying degrees. Figure 1 In the middle AE), all indicators of semen quality of R33, R36, R39, and R47 were not significantly different from or significantly better than F0.

[0029] Immunofluorescence detection of blood-testis barrier-related proteins, such as Figure 2 As shown, the ZO-1 membrane localization of F15 cells was severely disrupted, and the fluorescence signal was disordered, reflecting impaired tight junction integrity. This disruption may adversely affect the barrier function of the spermatogenic microenvironment. After 36 days of resumed feeding, ZO-1 cells re-anchored to the cell membrane, the tight junction structure was partially repaired, and the continuity of membrane signaling was improved.

[0030] Analysis of proteomic data from F15 vs F0, F15 vs R36, and R36 vs F0 revealed 29 significantly downregulated and 62 significantly upregulated proteins in F15 vs F0, 104 downregulated and 123 upregulated proteins in F15 vs R36, and a relatively smaller number of differentially regulated proteins in the R36 vs F0 group, including 19 downregulated and 30 upregulated proteins. Figure 3 KEGG pathway enrichment analysis was performed on DEPs from different comparison groups. The differentially expressed proteins in the F15 vs F0 group were significantly enriched in pathways such as Chemical carcinogenesis, SNARE interactions in vesicular transport, and Apoptosis. Figure 4 The differentially expressed proteins in the F15 vs R36 group were mainly enriched in cardiovascular disease-related pathways such as Hypertrophic Cardiomyopathy (HCM) and Arrhythmogenic Right Ventricular Cardiomyopathy (ARVC). Figure 4 (Middle B); for the R36vsF0 group, the differentially expressed proteins were mainly enriched in metabolic pathways such as Metabolic pathways, Glycosphingolipid metabolism, N-Glycan biosynthesis, and Renin-angiotensin system. Figure 4 (C)

[0031] Analysis of the phosphorylated protein data from three groups—F15 vs F0, F15 vs R36, and R36 vs F0—clarified the quantitative distribution of differentially phosphorylated proteins. Figure 5 In the F15 vs F0 group, 30 significantly downregulated proteins and 32 significantly upregulated proteins were detected; in the F15 vs R36 group, 33 downregulated proteins and 51 upregulated proteins were detected; and in the R36 vs F0 group, 12 downregulated proteins and 3 upregulated proteins were detected. This indicates that F15, compared to F0 and R36, regulates protein phosphorylation more broadly, leading to a more significant reshaping of the phosphorylation profile. KEGG enrichment analysis of differentially phosphorylated proteins showed that the differentially phosphorylated proteins in the F15 vs F0 group were significantly enriched in pathways such as the Renin-angiotensin system and Adherens junction. Figure 6 (A); the differentially expressed proteins in the F15 vs R36 group were mainly enriched in metabolic pathways such as the insulin signaling pathway and Type II diabetes mellitus. Figure 6 (B); the differentially expressed proteins in the R36vsF0 group were mainly enriched in pathways such as Arginine and proline metabolism and Pyruvate metabolism. Figure 6 (C)

[0032] There are 821 differentially expressed molecules in the proteome and phosphorylated proteome, some of which are regulated by both expression level and phosphorylation modification. Figure 7 In the comparison of F15 vs F0 and F15 vs R36 in the phosphoproteomics, a total of 212 differentially expressed molecules were found. Figure 7 KEGG enrichment analysis showed that differentially expressed molecules were significantly enriched in pathways such as Ribosome, Spliceosome, Apoptosis, and Tightjunction. Figure 7 The enrichment of C in the Tight junction may suggest that phosphorylation plays an important role in maintaining the blood-testis barrier.

[0033] By summarizing and organizing the omics data, it was found that in F15, the expression of differentially phosphorylated proteins increased slightly, but the abundance of differentially phosphorylated proteins and the ratio of phosphorylated protein abundance to protein abundance decreased. Figure 8 (AC). Further detailed analysis revealed increased expression of ZO-1 in F15, with increased phosphorylation abundance and the phosphorylation abundance / protein abundance ratio. In particular, two new phosphorylation sites, ZO-1 Ser1573 and Ser1620, were observed. Figure 8(DF). To investigate whether ZO-1 serine phosphorylation can respond to energy shortage in cells, starting from the expression and localization of ZO-1, compared with 10% serum treatment, the protein level of ZO-1 was upregulated at 1h-5%, 1h-7.5%, 12h-0%, 12h-1%, 24h-5%, 24h-7.5%, 48h-0%, 48h-1%, and 48h-5%. Figure 9 (A, B). IP values ​​of ZO-1 and P-Ser showed increased P-Ser / ZO-1 expression at 1h-1%, 1h-5%, 1h-7.5%, 3h-0%, 3h-1%, 3h-7.5%, 12h-0%, 12h-2.5%, 12h-5%, 12h-7.5%, 24h-0%, 24h-1%, 24h-2.5%, and 24h-5%. Figure 10 Based on Western blotting (WB) and intracellular injection (IP), treatment of Supporting Cells with different concentrations of serum for 1 h, 12 h, and 24 h increased ZO-1 protein expression and phosphorylation levels, consistent with sequencing results. Subsequent IP results showed that compared to 10% serum treatment, 5% serum treatment for 12 h increased ZO-1 expression and serine phosphorylation levels, but decreased these levels after 24 h of treatment. Figure 11 AC (Chinese)

[0034] Therefore, this invention, through proteomics and phosphoproteomics analysis of fasting-induced rooster testicular tissue, combined with bioinformatics screening, identified the Ser1573 and Ser1620 phosphorylation sites of ZO-1 as specific biomarkers for fasting-induced blood-testis barrier remodeling in chickens. This provides key molecular markers for assessing rooster testicular function and optimizing feed restriction programs in breeding roosters, and has significant value for production applications.

[0035] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A phosphorylated protein marker for fasting-induced remodeling of the blood-testis barrier in chickens, characterized in that, The biomarkers include the Ser1573 phosphorylation site and / or the Ser1620 phosphorylation site of the tight junction protein ZO-1.

2. The method for screening phosphorylated protein markers for fasting-induced blood-testis barrier remodeling in chickens as described in claim 1, characterized in that, Includes the following steps: S1. Sample Collection: Healthy adult "Yufen No. 1" H-strain roosters with uniform weight were selected. The experiment was divided into two phases: a pre-trial period and a regular feeding period. The regular feeding period included a fasting period and a recovery feeding period. The fasting period was divided into two stages: fasting and water restriction, and fasting and water restriction treatment. The recovery feeding period was also divided into two stages: first, a gradual transition with quantitative feeding and water, and then a return to free feeding and drinking. Testicular tissue samples were collected from the roosters at key time points during the experiment. S2. Protein extraction and enzymatic hydrolysis: Total protein was extracted from testicular tissue, subjected to reduction alkylation treatment, and then hydrolyzed with trypsin to obtain peptides. S3. Enrichment of phosphorylated peptides: Phosphorylated peptides were enriched using TiO2 affinity chromatography. S4. Proteomics and phosphoproteomics analysis: Liquid chromatography-tandem mass spectrometry was used to detect peptides and phosphorylated peptides to obtain protein expression levels and phosphorylation modification data; S5. Differential Analysis: By comparing and analyzing differences in protein expression and phosphorylation modification using bioinformatics software, significantly different phosphorylated proteins and sites are screened out. S6. Functional Validation: Western blot and immunofluorescence techniques were used to verify the expression and localization changes of differentially phosphorylated proteins. Combined with IP assays and cellular and molecular methods, specific biomarkers related to blood-testis barrier remodeling were identified.

3. The screening method according to claim 2, characterized in that, In S1, after feeding resumed, the sperm volume, sperm motility, sperm density, sperm abnormality rate, and effective sperm count were continuously measured to evaluate the remodeling of reproductive performance.

4. The screening method according to claim 2, characterized in that, In S5, bioinformatics analysis includes differentially expressed proteins, differentially phosphorylated proteins, and KEGG pathway enrichment.

5. The application of the phosphorylated protein marker as described in claim 1 in assessing the state of testis barrier remodeling in chicken blood, characterized in that, The Ser1573 phosphorylation site, Ser1620 phosphorylation site, and overall phosphorylation level of ZO-1 in testicular tissue were detected to determine whether the blood-testis barrier was intact.

6. The application of the phosphorylated protein marker as described in claim 1 in optimizing the feed restriction program for breeding roosters, characterized in that, Detecting the phosphorylation level of biomarkers helps to dynamically adjust fasting duration and refeeding nutrient ratio, thereby maintaining stable blood-testis barrier function.

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