Goat X and Y sperm sorting liquid, sorting reagent and sorting method

Abstract

The invention relates to the technical field of sperm sorting, in particular to a goat X and Y sperm sorting solution, a sorting reagent and a sorting method. The X and Y sperm sorting liquid takes water as a solvent, and comprises the following components by concentration: 24-27 g / L of glucose, 15-18 g / L of citric acid, 14-17 g / L of sodium ethylene diamine tetracetate, 55-59 g / L of sodium chloride, 19-22 g / L of sodium bicarbonate, 32-35 g / L of potassium chloride, 5.6-6.0 g / L of Tris, 0.4-0.8 g / L of BSA and 1.0 [mu] mol / L of requinimod R848. The R848 regulates and controls the movement performance of the goat X sperms, so that not only is the molecular effect of the R848 on an interaction network and phosphorylation modification of X sperm metabolites clarified, but also a brand new thought and theoretical basis are provided for precise optimization and safety evaluation of a sperm sorting technology.

Description

Technical Field

[0001] This invention relates to the field of sperm sorting technology, and more particularly to a goat X and Y sperm sorting solution, sorting reagent, and sorting method. Background Technology

[0002] With the development and application of modern advanced biotechnology, the production level of animals has been continuously improved. Some economic traits of animals are closely related to their sex, such as milk production, carcass weight, and egg production. In order to obtain offspring of the desired sex, sex control at the sperm and embryo stages is currently the main research direction of scientists. Among them, artificial insemination and in vitro fertilization using sex-controlled semen can give full play to the breeding value of male animals and the reproductive performance of female animals, which has a positive impact and important role in the development of the livestock industry.

[0003] Flow cytometry has improved the efficiency and accuracy of X and Y sperm separation in animals, and is currently the primary method for commercial production of sex-controlled semen internationally. With advancements in scientific research, researchers have improved the flow cytometer nozzles, electric fields, and other systems, and by adjusting voltage, have further enhanced separation efficiency. However, the commercial application of flow cytometry for sex-controlled semen production is currently limited to dairy cows, and its application in sheep, pigs, and other livestock remains difficult. X / Y semen sorting technology based on the TLR7 / 8 agonist R848 has become a simple and cost-effective method for sex control, showing potential for widespread application in livestock production. However, the R848 sorting technology still suffers from low sorting efficiency, and the effects of R848 on goat X sperm lack systematic and in-depth research, making it impossible to develop targeted molecular-level formulation optimization schemes suitable for "sorting-recovery-promotion" of this sorting method.

[0004] Based on this, the present invention is proposed. Summary of the Invention

[0005] This invention addresses the theoretical gap in TLR7 / 8 activation sorting systems by utilizing sequencing omics analysis to examine the inhibitory effect of remiquintimod R848 (R848) on the motility of goat X sperm, thereby clarifying the molecular regulatory mechanism of R848 on goat X sperm and providing molecular target information for targeted optimization of goat X and Y sperm formulations. Specifically, it relates to a goat X and Y sperm sorting solution, sorting reagent, and sorting method.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a goat X and Y sperm sorting solution, using water as a solvent and comprising the following components at the following concentrations: Glucose 24–27 g / L, citric acid 15–18 g / L, sodium EDTA 14–17 g / L, sodium chloride 55–59 g / L, sodium bicarbonate 19–22 g / L, potassium chloride 32–35 g / L, Tris 5.6–6.0 g / L, BSA 0.4–0.8 g / L, and requimomod R848 1.0 μmol / L.

[0007] This invention also provides the application of the aforementioned goat X and Y sperm sorting solution in the preparation of reagents for efficient sorting of goat X and Y sperm.

[0008] The present invention also provides a goat X and Y sperm sorting reagent, comprising goat X and Y sperm sorting solution, washing solution and requimomod R848; The goat X and Y sperm separation solution is the goat X and Y sperm separation solution; The washing solution, with water as the solvent, comprises the following components at the following concentrations: Tris 180~220 mM / 100mL, citric acid 70~90 mM / 100mL, and fructose 60~70 mM / 100mL.

[0009] This invention also provides the application of the aforementioned goat X and Y sperm sorting reagent in the efficient sorting of goat X and Y sperm.

[0010] This invention also provides a method for sorting X and Y sperm from goats, comprising the following steps: (1) Prepare each component according to the goat X and Y sperm sorting reagent described above; (2) Mix fresh goat semen with goat X and Y sperm separation solution, centrifuge, discard the supernatant, and obtain semen precipitate; (3) Mix the semen precipitate with the goat X and Y sperm sorting solution and adjust the sperm density to 5~7×10⁻⁶. 8 cells / mL, to obtain mixed semen; (4) Add Requimod R848 to the mixed semen at a final concentration of 0.7~0.9 μmol / L, incubate for 18~22 min, discard the upper layer of semen, take the lower layer of semen and mix it with the goat X and Y sperm sorting solution, add Requimod R848 at a final concentration of 0.5~0.7 μmol / L, incubate for 8~12 min, discard the upper layer of semen, take the lower layer of semen and mix it with the goat X and Y sperm sorting solution, centrifuge, discard the supernatant, and take the precipitate; resuspend the precipitate with washing solution to obtain goat X sperm; Add 0.3–0.5 μmol / L of Requimod R848 to the mixed semen and incubate for 28–32 min. Collect the supernatant. Mix the supernatant with goat X and Y sperm sorting solution, add 0.1–0.3 μmol / L of Requimod R848, and incubate for 9–11 min. Collect the supernatant and mix with the goat X and Y sperm sorting solution, centrifuge, discard the supernatant, and collect the precipitate. Resuspend the precipitate in washing solution to obtain goat Y sperm. In step (2), the centrifugation speed is 600×g~800×g, and the centrifugation time is 4~6 min; In step (4), the centrifugation speed is 700×g~900×g and the centrifugation time is 4~6 min.

[0011] This invention also provides the application of the method in the efficient sorting of goat X and Y sperm.

[0012] This invention also provides the application of requimituvir R848 as a sorting agent for goat X and Y sperm in inhibiting the normal motility of goat X sperm.

[0013] This invention also provides the application of requimituvir R848 as a sorting agent for goat X and Y sperm in interfering with the normal motility of goat X sperm.

[0014] Preferably, retinomod R848 inhibits or interferes with the normal motility of goat X sperm by downregulating the expression levels of PI4K2A and PI4K2B proteins in goat X sperm and altering their subcellular localization, while increasing the phosphorylation level of PTEN protein.

[0015] The present invention also provides the application of the expression level of PI4K2A protein, the expression level of PI4K2B protein, or the phosphorylation level of PTEN protein as targets in the preparation of reagents to improve abnormal motility of goat X sperm. The abnormal movement of goat X sperm was caused by the separation of goat X and Y sperm by requitomod R848 as a sorting reagent.

[0016] The present invention has the following advantages: This invention utilizes non-targeted metabolomics and targeted amino acid metabolomics techniques to comprehensively analyze the metabolite profiles of goat X sperm before and after R848 treatment. The results showed that R848 treatment led to significant changes in 380 metabolites in sperm, including key metabolites such as adenine, L-methionine, glutathione, and inositol. These metabolites were significantly enriched in 27 metabolic pathways, including glycine / serine / threonine metabolism, the tricarboxylic acid cycle, and purine metabolism, indicating that R848 affects the motility of X sperm by interfering with energy metabolism and the performance of the antioxidant system. Further phosphorylated proteomics analysis identified 1036 proteins with significantly upregulated phosphorylation levels and 410 proteins with downregulated levels. The expression and phosphorylation status of key signaling nodes such as PI4K2A, PI4K2B, and PTEN were significantly altered, suggesting that these proteins could serve as novel targets for the activation of the TLR7 / 8 signaling pathway and its influence on downstream phosphorylation networks.

[0017] This invention is the first to systematically apply multi-omics sequencing technology to elucidate the mechanism by which R848 regulates the motility of goat X sperm. It not only clarifies the molecular role of R848 in the interaction network of X sperm metabolites and phosphorylation modification, but also provides a new approach and theoretical basis for the precise optimization and safety assessment of sperm sorting technology. It provides a basis for the development of efficient sorting reagents for goat X and Y sperm, and provides a target for the recovery of abnormal circling movement of X sperm caused by R848 sorting. Attached Figure Description

[0018] Figure 1 Immunofluorescence staining images of key specific candidate proteins in fresh goat sperm (candidate proteins are TLR7, TLR8, GPR174, Eda2r, GPR34 and AR proteins; white arrows indicate positive sperm, yellow arrows indicate negative sperm, scale bar=10μm). Figure 2 A bar chart showing the proportion of positive sperm in fresh goat semen for key specific candidate proteins; Figure 3 The effect of different treatments on the proportion of upper-layer sperm in goats (vertical axis represents the proportion of upper-layer sperm). Figure 4 The effects of different treatments on the motility of goat sperm (left figure is the control group, right figure is the R848 treatment group). Figure 5 The effects of different treatments on the motility of lower sperm in goats; Figure 6 Analysis of acrosome and plasma membrane integrity of goat X sperm under different treatments (A and C are the results of acrosome integrity testing of goat X sperm; B and D are the results of plasma membrane integrity testing of goat X sperm; yellow arrows indicate abnormal sperm, white arrows indicate normal sperm, scale bar=10μm). Figure 7 Ultrastructural analysis of goat sperm (A is an electron micrograph of the ultrastructure, Bar=2.0μm; B~D are the structural integrity rates of different sperm segments); Figure 8 Analysis of energy metabolism levels in goat sperm (Figure A shows the ATP level detection in the lower layer of sperm; Figure B shows the mitochondrial membrane potential detection in the lower layer of sperm; Figure C shows the fluorescence spectrum of mitochondrial membrane potential in the lower layer of sperm (white arrows indicate red fluorescent polymers, yellow arrows indicate green fluorescent monomers, 100 μm)). Figure 9 To detect the separation effect of goat sperm after sorting by flow cytometry (Up represents upper layer sperm; Low represents lower layer sperm); Figure 10 To detect the separation efficiency of goat sperm after sorting using SRY immunofluorescence staining; Figure 11 To detect the separation effect of goat sperm after sorting using immunofluorescence staining (left image shows the proportion of X and Y sperm in the upper layer of sperm, right image shows the proportion of X and Y sperm in the lower layer of sperm); Figure 12 To detect the sperm count of goat sperm at different distances under different treatments using immunofluorescence staining; Figure 13 The following graphs show the statistical results of goat sperm count at different distances (left graph shows the number of goat sperm at 0.5cm; middle graph shows the number of goat sperm at 1.0cm; right graph shows the number of goat sperm at 1.5cm). Figure 14 A circular diagram showing the differential metabolite classification of goat sperm before and after sorting with requimomod R848; Figure 15 Pie chart showing superclass metabolite differences before and after sorting; Figure 16 Hierarchical clustering heatmap of metabolomes between groups before and after sorting of Requimod R848; Figure 17 PCA diagram of metabolome before and after sorting of requizimib R848; Figure 18 Volcano plot of differential metabolites before and after sorting of requizimib R848; Figure 19 Z-score of different metabolites before and after sorting of requimitudine R848; Figure 20 Hierarchical clustering diagram of differential metabolites before and after sorting of requimomod R848; Figure 21 Matchstick-shaped image showing the difference in metabolites of requizimide R848 before and after sorting; Figure 22Heatmap for correlation analysis of differential metabolites before and after sorting of requizimide R848; Figure 23 Chord diagram of differential metabolites before and after sorting of requimomod R848; Figure 24 Bubble plot of KEGG enrichment of differential metabolites of requimitubr R848 before and after sorting; Figure 25 A graph showing the abundance scores of metabolites before and after sorting of requizimide R848; Figure 26 Rectangular tree diagram of pathways enriched by differentially enriched metabolites before and after sorting of requimomod R848; Figure 27 A network diagram of the regulatory interactions of pathways for the enrichment of differential metabolites before and after requizimid R848 sorting; Figure 28 Analysis of amino acid target data before and after sorting of Requimod R848 (Figure A is PCA plot; Figure B is OPLS-DA plot; Figure C is OPLS-DA plot; Figure D is OPLS-DA model permutation test plot; Figure E is correlation analysis heatmap). Figure 29 A volcano plot showing the amino acid differences before and after sorting of requizimib R848; Figure 30 Z-score of amino acids differing before and after sorting of Requimod R848; Figure 31 Matchstick-shaped graph showing the top 10 amino acids with the most differences before and after sorting with quinomod R848; Figure 32 Hierarchical clustering diagram of amino acids related to differences in sperm motility function; Figure 33 Box plots showing the differences in amino acids before and after R848 sorting (top left: glycine box plot; top right: L-arginine box plot; bottom left: D-aspartic acid box plot; bottom right: L-cysteine ​​box plot). Figure 34 A graph showing the KEGG enrichment analysis of differential metabolites of quimomod R848 before and after sorting; Figure 35 Bubble diagram of metabolic pathways of differentially metabolized metabolites before and after sorting of quinomod R848; Figure 36 A graph showing the abundance scores of metabolites in key pathways before and after sorting of quinomod R848; Figure 37 SDS-PAGE images of phosphorylated proteins before and after sorting with quinomod R848; Figure 38 Chromatograms of phosphorylated proteins (TIC / BPC) before and after sorting with quinomod R848; Figure 39Basic statistical graph of mass spectrometry data of phosphorylated proteins before and after sorting with quinomod R848; Figure 40 The following are characteristic analysis diagrams of differentially phosphorylated modification sites before and after sorting of quinmod R848 (Figure A shows the distribution of the number of phosphorylated modification sites; Figure B shows the distribution of peptide length; Figure C shows the distribution of the number of phosphorylated sites in upregulated proteins; Figure D shows the distribution of the number of phosphorylated sites in downregulated proteins; Figure E shows the percentage of amino acid modification types at differentially phosphorylated sites). Figure 41 Figures showing the repeatability verification of quinmod R848 samples before and after sorting and the screening analysis of differentially phosphorylated peptides (Figure A: Pearson correlation coefficient; Figure B: principal component analysis; Figure C: principal component score; Figure D: hierarchical cluster analysis; Figure E: volcano plot of differentially phosphorylated peptides). Figure 42 Top 10 GO enrichment analysis classification bar chart of differentially phosphorylated proteins before and after quimod R848 sorting; Figure 43 Classification bar chart of GO enrichment analysis of sub-motor correlation before and after sorting of quintimod R848; Figure 44 Bubble plot of KEGG metabolic pathway enrichment for differentially phosphorylated proteins before and after R848 sorting; Figure 45 Top 10 bubble chart of KEGG metabolic pathway enrichment of differentially phosphorylated proteins before and after R848 sorting; Figure 46 A bar chart for COG analysis of differentially expressed peptide genus proteins; Figure 47 Analysis of differentially phosphorylated motif information (left figure is the enrichment fraction of differentially phosphorylated motifs; right figure is the frequency of phosphorylated modified peptides corresponding to differentially phosphorylated motifs). Figure 48 A visualization of differential phosphorylation motifs; Figure 49 Bubble chart analysis for the TOP 25 structural domains; Figure 50 Subcellular structural localization of differentially phosphorylated proteins before and after quinomod R848 sorting; Figure 51 A network diagram of differentially phosphorylated protein interactions before and after quinomod R848 sorting; Figure 52 A bar chart showing the frequency of differentially phosphorylated protein interactions before and after quinomod R848 sorting; Figure 53 A diagram illustrating the interaction between PPIs, key targets of differentially phosphorylated proteins before and after quinomod R848 sorting; Figure 54Map of TLR7 / CD4 protein docking sites (left image shows molecular docking, right image shows highlighted data); Figure 55 Map of TLR8 / CD4 protein docking sites (left image shows molecular docking, right image shows highlighted data); Figure 56 Map of CD4 / PTEN protein docking sites (left image shows molecular docking, right image shows highlighted area); Figure 57 Map of PI4K2A / PTEN protein docking sites (left image shows molecular docking, right image shows highlighted area); Figure 58 Diagram of PI4K2B / PTEN protein docking sites (left image shows molecular docking, right image shows highlighted area); Figure 59 Map of PIP5K1A / PTEN protein docking sites (left image shows molecular docking, right image shows highlighted area); Figure 60 Western blot analysis was performed to investigate the effects of quimomod R848 on key proteins in the TLR7 / 8 signaling pathway. Figure 61 To detect the effect of quimod R848 on key proteins in the TLR7 / 8 signaling pathway using immunofluorescence staining; Figure 62 To analyze the effects of quinomod R848 on key proteins in the TLR7 / 8 signaling pathway using flow cytometry. Detailed Implementation

[0019] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0020] The remiquimod R848 described in this embodiment was purchased from Novus Biologicals (Littleton, CO, USA); all reagents except remiquimod R848 in this embodiment were purchased from Sigma-Aldrich.

[0021] Example 1

[0022] X and Y sperm sorting from goats

[0023] Preparation of X and Y goat sperm separation solution: Add 25 g glucose, 17 g citric acid, 15 g sodium EDTA, 57 g sodium chloride, 20 g sodium bicarbonate, 33 g potassium chloride, 5.8 g Tris, 0.6 g BSA and 1.0 μmol requimide R848 to 1 L of water and mix thoroughly.

[0024] Preparation of washing solution: Tris 200 mM, citric acid 80 mM, fructose 65 mM, ultrapure water 100 mL, mix well.

[0025] Sorting method: (1) Mix 0.5 mL of fresh semen from Guanzhong dairy goats with 3 mL of goat X and Y sperm separation solution, centrifuge at RCF 700×g for 5 min, discard the supernatant and keep the precipitate, and remove the supernatant seminal plasma as completely as possible; (2) The obtained semen precipitate was resuspended in goat X and Y sperm sorting solution preheated at 37℃, and its sperm density was adjusted to 6×10⁻⁶. 8 The concentration of cells / mL was aliquoted into 15 mL centrifuge tubes at a volume of 3 mL to obtain a mixed semen. (3) Starting from this step, X sperm / Y sperm separation is performed. Separation of X sperm: During the first incubation, add 0.8 μmol / L retinoic acid R848 and incubate for 20 min. Discard the supernatant (2 mL) and transfer the remaining 1 mL to a new 15 mL centrifuge tube. During the second incubation, add 2 mL of goat X and Y sperm separation medium to a volume of 3 mL, add 0.6 μmol / L retinoic acid R848 and incubate for 10 min. Discard the supernatant (2 mL) and transfer the remaining 1 mL to a new 15 mL centrifuge tube. Add 2 mL of goat X and Y sperm separation medium to a volume of 3 mL. Elute twice using a centrifuge at RCF 800×g for 5 min. Discard the supernatant and retain the precipitate to obtain goat X sperm. Resuspend the precipitate in washing buffer until the sperm density reaches 6×10⁻⁶. 8 cells / mL to be used.

[0026] Separation of Y sperm: During the first incubation, add 0.4 μmol / L retinoic acid R848 and incubate for 30 min. Transfer 1 mL of the supernatant to a new 15 mL centrifuge tube. During the second incubation, add 2 mL of goat X and Y sperm separation medium to a final volume of 3 mL, add 0.2 μmol / L retinoic acid R848 and incubate for 10 min. Transfer 1 mL of the supernatant to a new 15 mL centrifuge tube, add 2 mL of goat X and Y sperm separation medium to a final volume of 3 mL, and centrifuge twice using RCF 700×g for 5 min. Discard the supernatant and retain the precipitate to obtain goat Y sperm. Resuspend the precipitate in washing buffer until the sperm density is 6×10⁻⁶. 8 cells / mL to be used.

[0027] Example 2

[0028] Immunofluorescence staining analysis of candidate proteins

[0029] (1) Smearing: Spread 30 μL of fresh goat semen evenly on the adhesive slide and let it air dry naturally; (2) Fixation: After the slides have been air-dried, they were soaked in anhydrous methanol for 10 min (methanol was recovered) and then air-dried naturally; they were washed with PBS 3 times for 5 min each time. (3) Permeation: After shaking off the PBS, add 200 μL of 0.5% Triton-100 evenly to the slide and permeate at room temperature for 30 min; wash with PBS 3 times, 5 min each time; (4) Blocking: After the PBS is spun dry, 200 μL of 5% BSA is evenly added to the glass slide and incubated at 37℃ for 30 min for blocking; (5) Add primary antibody: Gently shake off the blocking solution, drop the primary antibody (TLR7 / TLR8 / GPR174 / Eda2r / GPR34 / AR / GPR174 antibody) diluted with PBST containing 1% BSA onto the glass slide, place the glass slide flat in the humidified chamber, and incubate overnight at 4°C. (6) Adding secondary antibody: Place the slide in PBS and wash it three times on a decolorizing shaker for 5 min each time. Shake the slide slightly to dry it and add the secondary antibody (cy3 or FITC fluorescent secondary antibody homologous to the primary antibody) diluted in PBST containing 1% BSA. Incubate at 37°C in the dark for 1 h. Then place the slide in PBS and wash it three times on a decolorizing shaker for 5 min each time. (7) After the sections are slightly dried, DAPI staining solution is added to the slide and incubated at room temperature in the dark for 10 min. (8) Autofluorescence quenching: Place the slide in PBS and wash it three times on a decolorizing shaker for 5 min each time. After slightly drying the slide, add autofluorescence quenching agent to the circle for 5 min, and rinse with PBS for 10 min; (9) Mounting: After slightly drying the sections, mount them with anti-fluorescence quenching mounting medium. Acquire images under a fluorescence microscope.

[0030] The results are as follows Figures 1-2As shown, TLR7 and TLR8 proteins served as controls, with positive sperm expression rates of 47.22%±2.93% and 48.08%±3.42%, respectively. TLR7 was primarily located at the sperm terminal, while TLR8 was primarily located in the sperm midpiece. GPR174 protein was mainly located in the sperm head, with a positive sperm expression rate of 50.95%±2.04%. Eda2r protein was mainly located on both sides of the acrosome, with a positive sperm expression rate of 50.58%±4.01%. GPR34 and AR proteins were not expressed in goat sperm. These results indicate that, except for TLR7 and TLR8, GPR174 and Eda2r proteins are partially expressed in goat sperm, and their expression rates are consistent with the theoretical wild-type X / Y sperm ratio, making them potential specific candidate proteins for X / Y sperm separation.

[0031] Figure 2 + indicates positive, - indicates negative, and the vertical axis represents the sperm percentage.

[0032] Example 3

[0033] Sperm motility trajectory and motility performance testing

[0034] (1) Place the glass slide and coverslip on a 37°C constant temperature table for preheating; (2) Take 10 μL of goat semen and drop it onto a preheated glass slide. Cover it with a coverslip and let it stand for a while. Then use the CASA system to detect the sperm motility trajectory and sperm motility performance. (3) The movement trajectories of the upper and lower layers of sperm in the mixed semen of the group without added remiquimod R848 and Example 1, as well as the lower layer sperm after elution (goat X sperm in Example 1), were captured at a frequency of 60 Hz under a 20x microscope using the CASA system (HVIEW-SSA V8.0). The sperm motility rate, sperm motility, average strain line velocity, average path velocity, average curve line velocity, linearity (LIN), progressiveness (STR), and wobble (WOB) were detected. (4) Select at least 5 clear fields of view, each containing at least 200 sperm, and then perform data analysis.

[0035] The results are as follows Figures 3-5As shown, after incubating goat semen with retinoic acid R848, the proportion of sperm in the upper third was significantly lower than that in the control group. The results indicate that retinoic acid R848 can induce stratification of goat sperm, which can be used for further research. The sperm motility trajectories in the lower layers of the control group and the retinoic acid R848 group were captured using CASA. The results showed that the sperm motility trajectories in the lower layer of the control group were longer and mostly linear, while retinoic acid R848 caused the lower layer sperm to move in circles and their trajectories to shorten (white arrows indicate linearly moving sperm; yellow arrows indicate circling sperm). Further investigation and analysis of the effects of retinoic acid R848 on the sperm motility and movement parameters in the lower layer were conducted. Figure 5 As shown in the results, the motility of the lower sperm cells (X sperm) in the requimitudine R848 treatment group was significantly reduced compared to the control group. Furthermore, analysis of sperm kinetic parameters revealed that, compared to the control group, requimitudine R848 significantly reduced the linearity (LIN), progressiveness (STR), linear velocity (VSL), curvilinear velocity (VCL), average path velocity (VAP), wobble (WOB), and lateral wobble amplitude (ALH) of the lower sperm cells. However, the movement angle (MAD) of the lower sperm cells in the requimitudine R848 treatment group was significantly increased compared to the control group; there was no significant difference in whiplash frequency (BCF) between the two groups.

[0036] Example 4

[0037] Goat sperm acrosome integrity testing

[0038] Sperm were stained and examined under a microscope using a fluorescent dye called fluorescein isothiocyanate-peanut lectin (FITC-PNA).

[0039] (1) Take 30 μL of the lower layer sperm sample obtained by sorting in Example 1 and spread it evenly on a special poly-lysine-treated adhesive glass slide, and let it air dry naturally in a black humid box; (2) After air drying, the slides were fixed in anhydrous methanol for 10 min, and then placed in the air to air dry naturally; (3) Take 30 μL of FITC-PNA working solution and drop it evenly onto the sample slide to ensure that the sample is covered. Then place the slide in a black humidified box and incubate it in a constant temperature oven at 37℃ for 30 min in the dark. (4) Take 30 μL of DAPI staining solution and evenly drop it onto the sample slide to ensure that the sample is covered. Place the slide in a black humidified box and incubate at room temperature for 8 min for staining. (5) After incubation, rinse the slide twice with PBS to remove the floating color. After air drying, add an appropriate amount of glycerol, seal and cover with a coverslip. Observe and take pictures under a fluorescence microscope. Sperm with complete bright green fluorescence at the front of the acrosome are sperm with complete acrosomes. Sperm with incomplete or no fluorescence at the front of the acrosome are sperm with damaged acrosomes. (6) When taking photos, at least 5 clear fields of view must be selected, and each field of view must contain at least 200 sperm. The entire experiment must be conducted in the dark. (7) After DAPI staining, the sperm cell nuclei fluoresce blue under a fluorescence microscope (DAPI fluorescent dye is used to stain the sperm cell nuclei to eliminate the influence of false positives caused by impurities and other non-cellular substances on sperm count).

[0040] Figure 6 The results show that there was no significant difference between the group treated with no retinoic acid R848 and the group treated with retinoic acid R848.

[0041] Example 5

[0042] Goat sperm plasma membrane integrity test

[0043] Sperm were stained and examined under a microscope using SYBR-14 / PI fluorescent dye.

[0044] (1) Take 200 μL of X and Y semen samples obtained by sorting in Example 1 and place them in a 1.5 mL centrifuge tube and incubate in a water bath at 37 °C for 10 min; (2) Add 0.2 μL of SYBR-14 working solution, shake gently to mix, and incubate in a 37℃ incubator for 8 min; (3) Add 1 μL of PI working solution, shake gently to mix, and incubate in a constant temperature incubator at 37℃ for 8 min; (4) After incubation, take 10 μL of semen sample and drop it onto a glass slide treated with poly-L-lysine. Cover with a coverslip and quickly place it under a fluorescence microscope to take a picture. (5) When taking photos, at least 5 clear fields of view must be selected, and each field of view must contain at least 200 sperm; the entire experiment must be conducted in the dark.

[0045] Figure 6 The results show that there was no significant difference between the group treated with no retinoic acid R848 and the group treated with retinoic acid R848.

[0046] Example 6

[0047] Ultrastructure detection of goat sperm

[0048] (1) Collect the test semen (the lower part of the mixed semen after sorting or the X sperm obtained by separation) by centrifugation and resuspend it at 4°C with electron microscopy fixative, mix well and fix for 3h; (2) Centrifuge, discard the supernatant, add 0.1 M phosphate buffer PBS (pH 7.4), mix well, rinse for 3 min, and then centrifuge again. Repeat 3 times. (3) Fix 1% osmium tetroxide prepared with 0.1 M PBS (pH 7.4) at room temperature in the dark for 2 h, and then rinse 3 times (15 min / time) with 0.1 M phosphate buffer PB (pH 7.4). (4) The samples were dehydrated for 15 minutes each time using 50%, 70%, 80%, 90%, 95%, and 100% acetone, respectively. (5) The samples were placed in acetone solutions of 5:1, 3:1, 1:1, 1:3, and 1:5 and infiltrated overnight at 37°C. Then the samples were inserted into the embedding plate and baked overnight at 37°C. Finally, the embedding plate was placed in a 60°C environment for polymerization for 48 h. (6) Slice the material at 70 nm using an ultrathin slicer and retrieve it using a 150-mesh copper mesh screen; (7) Stain copper mesh in 2% uranium acetate saturated alcohol solution in the dark for 8 min; wash 3 times with 70% alcohol; wash 3 times with ultrapure water; stain 8 min in 2.6% lead citrate solution in the dark; wash 3 times with ultrapure water, and blot dry with filter paper. Place copper mesh sections in a copper mesh box and dry at room temperature overnight.

[0049] Observe under a transmission electron microscope and collect and analyze images.

[0050] Figure 7 The results shown in A indicate that the sperm mitochondrial cristae number was normal and the outer membrane was intact in both the group without Requimod R848 treatment (lower layer of fresh semen) and the group with Requimod R848. Figure 7 The midpiece, principal piece, and terminal piece structures of sperm from groups B to D showed normal structures, with no significant differences between the two groups. Ultrastructural analysis of sperm in both groups showed that the axonal filament of the flagellum was surrounded by a fibrous sheath, and the central microtubules and inner and outer dynein arms were intact.

[0051] Example 7

[0052] ATP content detection in goat sperm

[0053] The ATP content of sperm was detected and analyzed using an ATP assay kit (S0026, Shanghai Beyotime Biotechnology Co., Ltd.). The specific operation and analysis are as follows: (1) Mix the ATP test reagent with the ATP test reagent dilution solution (1:9) and store on ice; (2) Take 1 mL of the lower semen of the group without added requitolimide R848 (lower semen of fresh semen) and 1 mL of the lower semen after sorting treated with requitolimide R848 in Example 1. Wash with PBS (2×, 10 min); add ATP lysis buffer (200 μL), and then lyse the sperm using a centrifuge at 4 ℃ (12000 g, 10 min). Take the supernatant for testing (operate on ice). (3) Add 100 μL of ATP detection working solution to a 96-well plate, let it stand at room temperature for 4 min, then add the supernatant to be tested (20 μL / well) and let it stand for 2 s. Finally, use an ELISA reader to shake and mix the sample and measure the luminescence value of the Luminometer, and calculate the sperm ATP level according to the instructions.

[0054] Figure 8 The results show that Requimod R848 can significantly reduce the ATP level in the lower sperm cells of goats.

[0055] Example 8

[0056] Goat sperm MMP level detection

[0057] Sperm mitochondrial membrane potential was detected using the JC-1 mitochondrial membrane potential detection kit (M8650, Beijing Solarbio Science & Technology Co., Ltd.). Specific procedures and analysis are as follows: (1) Take 1 mL of the lower semen of the group without added requimide R848 (fresh semen lower semen), the control group and the lower semen after sorting treated with requimide R848 in Example 1, and resuspend them in 0.5 mL PBS for testing; (2) Mix 50 μL JC-1 (200×) with 8 mL of ultrapure water and shake, then add JC-1 staining buffer (5×, 2 mL) to prepare JC-1 staining working solution for later use; (3) Mix 1 mL of JC-1 staining buffer (5×) with 4 mL of distilled water by inverting and place on ice for later use; (4) Add JC-1 staining working solution to the semen to be tested, mix by inversion and incubate at 37 °C for 20 min; then centrifuge at 600 g for 3 min at 4 °C, discard the supernatant, add 1 mL of JC-1 staining buffer to the precipitate to resuspend, centrifuge at 600 g for 3 min at 4 °C again, repeat 3 times and discard the supernatant. (5) Resuspend an appropriate amount of JC-1 staining buffer (1×) and analyze using a microplate reader. When detecting JC-1 monomer, the excitation light is set to 490 nm and the emission light is set to 530 nm; when detecting JC-1 polymer, the excitation light is set to 525 nm and the emission light is set to 590 nm. Finally, calculate the mitochondrial membrane potential level according to the instructions of the mitochondrial membrane potential detection kit.

[0058] Figure 8 The results show that sperm MMP levels were significantly lower in the Requimod R848 group compared to the control group.

[0059] Example 9

[0060] Flow cytometry analysis of sperm X / Y ratio

[0061] Sperm staining with Hoechst 33342 fluorescent dye was performed using flow cytometry. The specific steps are as follows: (1) Take 500 μL of the sorted mixed semen from Example 1 and place it in a 1.5 mL centrifuge tube (wrapped in aluminum foil); (2) Add 8–15 μL of Hoechst 33342 fluorescent dye according to different sperm densities; (3) Sperm density maintained at 2×10 8 cells / mL, incubated in a 37°C water bath for 50 min in a dark environment; (4) Based on the degree of nuclear staining, the DAPI channel with an excitation wavelength of 355 nm was selected in the flow cytometer for detection, and the proportion analysis was performed using FlowJo-V10 according to the sperm clustering.

[0062] Figure 9 The results show that after sorting with requizimib R848, the proportion of Y sperm in the upper layer of sperm was 89.1%, and the proportion of X sperm was 8.56%; while in the lower layer of sperm, the proportion of Y sperm was 22.9%, and the proportion of X sperm was 74.7%. Immunofluorescence staining of the upper and lower layers of sperm after requizimib R848 treatment revealed that SRY was expressed in the sperm tail; SRY-negative sperm (SRY-) were X sperm, and SRY-positive sperm (SRY+) were Y sperm. Figure 10 The results of immunofluorescence staining analysis are as follows: Figure 11 As shown, in the upper layer of sperm, SRY- sperm (X sperm) accounted for 9%±1.63%, and SRY+ sperm (Y sperm) accounted for 91%±1.63%; in the lower layer of sperm, SRY- sperm (X sperm) accounted for 87%±4.58%, and SRY+ sperm (Y sperm) accounted for 9.67%±2.08%.

[0063] Example 10

[0064] Sperm-cervical mucus model migration rate detection

[0065] (1) Semen sample pretreatment and staining: Take an appropriate amount of fresh semen sample, sorted X semen and Hoechst staining solution, mix thoroughly, and then incubate the mixture at room temperature for 10 min for staining. (2) Take a capillary tube with a length of 8 cm and an inner diameter of 0.8 mm. Insert one end of the capillary tube into the separated egg white and use the siphon principle to draw the egg white (or a standardized cervical mucus model) into the capillary tube, ensuring that there are no air bubbles in the tube. Use sealing powder or heating with an alcohol lamp to completely seal the suction end of the capillary tube as a sealed end; (3) Add 200 μL of stained semen sample to a clean 1.5 mL centrifuge tube, and insert the unsealed end of the capillary tube into the semen sample at a 45° angle. Then place the entire device in a constant temperature environment of 37°C and incubate for 30 min. (4) After incubation, carefully remove the capillary tube. Use a fluorescence microscope to observe and detect the penetration of sperm as they migrate forward within the capillary tube from the unsealed original end. Count the number of sperm in the field of view at 0.5 cm, 1.0 cm, and 1.5 cm to assess the sperm penetration efficiency.

[0066] Figures 12-13 The results show that, compared with the control group (fresh semen), the sperm count in the requimide R848 treatment group was not significantly different at 0.5 cm, but was significantly reduced at 1.0 and 1.5 cm.

[0067] Example 11

[0068] Non-targeted metabolomics detection of goat sperm

[0069] (1) Transfer the goat X sperm sample (lower layer of mixed semen) obtained in Example 1 to an EP tube and add 4 times the extraction solution (methanol:acetonitrile = 1:1 (V / V)), the extraction solution contains isotope-labeled internal standard; (2) Vortex mix for 30 s, sonicate for 10 min (ice water bath); let stand at -40 ℃ for 1 h; (3) Centrifuge the sample at 4 ℃, 12000 rpm (centrifugal force 13800 (×g), radius 8.6 cm) for 15 min; Collect the supernatant into a sample vial for instrumental analysis; (4) Take equal amounts of supernatant from all samples and mix them to form a QC sample for instrument testing; (5) For polar metabolites, this project used a Vanquish (Thermo Fisher Scientific) ultra-high performance liquid chromatograph and a Waters ACQUITY UPLC BEH Amide (2.1 mm × 50 mm, 1.7 μm) column to separate the target compounds. Phase A of the liquid chromatography was aqueous, containing 25 mmol / L ammonium acetate and 25 mmol / L ammonia, and phase B was acetonitrile; sample tray temperature: 4 ℃, injection volume: 2 μL; (6) The Orbitrap Exploris 120 mass spectrometer can perform primary and secondary mass spectrometry data acquisition under the control of the control software (Xcalibur, version: 4.4, Thermo). Detailed parameters are as follows: Sheath gas flow rate: 50 Arb, Aux gas flow rate: 15 Arb, Capillary temperature: 320℃, Full ms resolution: 60000, MS / MS resolution: 15000, Collision energy: SNCE 20 / 30 / 40, Spray Voltage: 3.8kV (positive) or -3.4 kV (negative); (7) After the raw data was converted into mzXML format by ProteoWizard software, metabolites were identified using a collaboratively developed R package. The database used was BiotreeDB (V3.0), and then visualization analysis was performed using a self-developed R package.

[0070] Figures 14-27As shown, a total of 21,989 features were retained after preprocessing, of which 1,240 were secondary qualitative substances. These substances can be divided into 23 superclasses, 125 classes, and 253 subclasses. Among the superclasses, lipids and lipid molecules accounted for the highest proportion (22.661%), followed by organic heterocyclic compounds (15.565%), while amino acids and peptides (2.419%), carbohydrates (1.048%), organic sulfur compounds (0.161%), and polyketides (0.484%) accounted for a relatively low proportion. Cluster heatmap and PCA analysis revealed that the metabolomics data of samples within the group had good repeatability and good clustering effect, and the differences between groups were significant. The separation of QC groups met the quality control expectations and effectively reflected the similarity and differences between samples. Using VIP>1 and P-value<0.05, fold change≥2 and fold change≤0.5 as screening criteria, a total of 380 differentially expressed metabolites were identified, of which 195 were upregulated (such as adenine, L-methionine, L-lysine, etc.) and 185 were downregulated (such as hypoxanthine, glutathione, inositol, etc.). Through Z-score analysis, cluster analysis, matchstick plot, correlation analysis and chord analysis, the differentially expressed metabolites were visualized and their synergistic regulatory relationships were analyzed, showing that the differentially expressed metabolites cover categories such as benzenes, lipids and lipid molecules, organic acids and their derivatives. KEGG pathway enrichment analysis revealed that differentially metabolites were significantly enriched in 27 metabolic pathways, including glycine-serine-threonine metabolism, alanine-aspartate-glutamate metabolism, purine metabolism, citric acid cycle, and pyruvate metabolism. Among these, pathways such as glycine-serine-threonine metabolism and phenylalanine metabolism were inhibited. Further pathway search and regulatory interaction network analysis clarified the intersection of sperm metabolic pathways and potential target enzymes and metabolites before and after treatment with retinomod R848.

[0071] Example 12

[0072] Targeted metabolomics detection of amino acids in goat sperm

[0073] (1) Thaw the sorted X sperm samples in an ice-water bath, and vortex for 30 s to mix before sampling;

[0074] (2) Accurately transfer 50 μL of sample into a 1.5 mL EP tube using a pipette, add 200 μL of extraction buffer (acetonitrile:methanol:volume ratio 1:1, mixed with isotope internal standard, pre-cooled at -40 ℃), and vortex mix for 30 s; (3) Ultrasound in an ice water bath for 15 min; (4) Let the sample solution stand at -40 ℃ for 1 h; (5) The sample was centrifuged at 4 ℃, 12000 rpm (centrifugal force 13800 (×g), radius 8.6 cm) for 15 min; (6) Take 100 μL of the supernatant and evaporate it to dryness by rotary evaporation; redissolve it with 100 μL of 50% methanol-water solution, add 100 μL of derivatizing agent, add 50 μL of 1 M NaHCO3, and vortex to mix; derivatize in a 40 ℃ water bath for 1 h, remove and cool to room temperature, add 50 μL of 2 M HCl, and evaporate to dryness by rotary evaporation; redissolve it with 200 μL of methanol and put it into the instrument; (7) Preparation of Standards: Accurately weigh the corresponding amount of standard into a 10 mL volumetric flask and prepare 10 mmol / L standard stock solutions. Take the corresponding amount of standard stock solution into a 10 mL volumetric flask and prepare a mixed standard solution. Dilute the standard solution sequentially to obtain a series of calibration solutions (containing a mixture of isotope-labeled internal standards with the same concentration as in the sample); (8) Mobile phase conditions: This project used a Thermovanquish UHPLC System (Thermo Fisher) for ultra-high performance liquid chromatography, employing a Waters ACQUITY UPLC BEH C18 (100×2.1 mm, 1.7 μm, USA) column for chromatographic separation of the target compounds. Phase A of the liquid chromatography consisted of 5 mM ammonium acetate, and phase B consisted of acetonitrile. The column oven temperature was 45℃, the sample pan temperature was 4℃, and the injection volume was 2 μL. Mass spectrometry conditions: This project used a ThermoAltis TSQPlus Mass Spectrometer triple quadrupole mass spectrometer, matched with an ESI electrospray ionization source, and performed mass spectrometry analysis in multiple reaction monitoring (MRM) mode. The ion source parameters are as follows: SprayVoltage=-3300 V, Sheath Gas=40 Arb, Aux Gas=10 Arb, Sweep Gas=1 Arb, Ion Transfer Tube Temp=325℃, Vaporizer Temp=350℃.

[0075] (9) Dilute the calibration solution by 2 times and perform UHPLC-MRM-MS analysis. Calculate the limit of detection and limit of quantitation of the method based on the signal-to-noise ratio. The limit of detection (LLOD) is defined as the compound concentration corresponding to a signal-to-noise ratio of 3, and the limit of quantitation (LLOQ) is defined as the compound concentration corresponding to a signal-to-noise ratio of 10 (US FDA guideline for bioanalytical method validation).

[0076] (10) During sample detection, the final concentration CF (Final Concentration, nmol / L) is the concentration CC (Calculated Concentration, nmol / L) directly measured by the instrument multiplied by the dilution factor Dil (Dilution Factor), with units of nmol / L; CM (Metabolite Concentration, nmol / L) is the concentration of the target metabolite in the sample, which is equal to the amount of the target metabolite in the sample CF multiplied by the final sample volume VF (Volume, μL), divided by the sample volume Vs (Volume, μL), with units of nmol / L. “NA” indicates that the target compound was not detected in the sample. “Red highlight” indicates that the concentration of the target metabolite in the sample is higher than the upper limit of quantitation (ULOQs), and “blue highlight” indicates that the concentration of the target metabolite in the sample is lower than the lower limit of quantitation (LLOQs), both indicating that the quantitative accuracy of the corresponding sample is relatively low.

[0077] Figures 28-36The results show that PCA, OPLS-DA, and PLS-DA analyses revealed a clear separation trend between the control group and the requimitudine R848 group, indicating good sample repeatability and data reliability. Furthermore, the established OPLS-DA model (R2Y=0.83, Q2Y=0.57) showed no overfitting issues. Pearson analysis of metabolite quantification values, presented in a heatmap, clarified the synergistic changes among metabolites. Based on the OPLS-DA results, differentially expressed amino acids were screened using the criteria of VIP>0 and P-value<1. Requimitudine R848 treatment significantly altered 63 amino acids, with 33 upregulated (e.g., glycine, L-alanine, L-arginine) and 30 downregulated (e.g., D-alanine, D-arginine, D-aspartic acid). Matchstick visualization of the top 10 upregulated and downregulated amino acids showed significant changes in D-histidine, D-arginine, and L-aspartic acid, suggesting strong activation / inhibition of their corresponding enzyme gene expression. Hierarchical clustering analysis of differentially expressed amino acids related to sperm motility showed high similarity in molecular modification patterns between the requitolimide R848 group and the control group, with significant differences between the groups. Box plot visualization of four key amino acids revealed that requitolimide R848 treatment significantly increased L-arginine content in sperm, while glycine, L-cysteine, and D-aspartic acid showed a decreasing trend, but the differences were not significant. KEGG pathway enrichment analysis showed that differentially expressed amino acids were enriched in 86 metabolic pathways, including amino acid biosynthesis, protein digestion and absorption, and amino acid acyl-tRNA biosynthesis. Among them, aminoacyl-tRNA biosynthesis was significantly enriched, which is the core pathway for requitolimide R848 to regulate sperm motility. Differential abundance analysis of key pathways showed significant differences in amino acid metabolism pathways (such as arginine biosynthesis) and membrane transport pathways (such as ABC transporters), suggesting that requitolimide R848 can simultaneously affect multiple functional modules such as "amino acid metabolism" and "membrane transport".

[0078] Example 13

[0079] Label-free quantitative proteomics detection of goat sperm phosphorylation

[0080] (1) Protein extraction: Take 1 / 2 of the sorted X sperm sample into a new EP tube, add 400 μL of 8 M Urea working solution and mix thoroughly. Sonicate on ice for 20 min, and let stand at 4 ℃ for 1 h to fully lyse the sample. Centrifuge at 12000 rpm and 4 ℃ for 10 min, and transfer the supernatant to a new EP tube; (2) Add 200 μL of BCA working solution to each well of a 96-well plate, for 7 standard spots and 1 blank. Add 20 μL of sample (diluted accordingly) or standard protein (BSA). Shake at 37 ℃ for 30 min and measure the absorbance at 562 nm. Fit a standard curve based on the standard protein and calculate the protein concentration of the corresponding sample; (3) Add TCEP to 5 mM and incubate at 55℃ with shaking for 30 min to reduce disulfide bonds. Cool the sample to room temperature, add CAA to 15 mM and react in the dark for 15 min to alkylate the reduced disulfide bonds; (4) Dilute 8 M MUrea to below 2 M with 100 mM HEPES, dissolve Trypsin in Resuspension buffer, incubate at room temperature for 5 min, and mix Trypsin with the sample at a ratio of Trypsin:protein = 1:100. After simple centrifugation, incubate overnight at 37 ℃ with shaking at 1000 rpm. (5) Add TFA to the mixed sample (final concentration 2%), mix thoroughly to precipitate SDC, centrifuge at high speed for 10 min, and transfer the supernatant to a new EP tube; (6) Activate the C18 column with 2 mL of Buffer C, allowing all the solution to slowly flow into the centrifuge tube. Add 2 mL of Buffer A to equilibrate, allowing all the solution to slowly flow into the centrifuge tube. Add the sample supernatant, allowing all the solution to slowly flow into the centrifuge tube, and collect the eluent (FT). Add 2 mL of Buffer A to rinse once, allowing all the solution to slowly flow into the centrifuge tube. Add Buffer B to elute, and use a pipette to press the eluent into a new EP tube and place it at -4°C overnight under vacuum to dry.

[0081] (7) Phosphorylated peptides were enriched according to the instructions of the High-Select™ TiO2 / IMAC PhosphopeptideEnrichment Kit (Thermo Fisher Scientific), and the enriched phosphorylated peptides were freeze-dried. (8) Approximately 200 ng of total peptides were taken from each sample and separated using a nano-UPLC liquid chromatography system (model: nanoElute2). The sample was then coupled with a mass spectrometer equipped with a nanoliter ion source (model: timsTOFPro2) for data acquisition. The chromatographic separation step used a 75 μm ID×25 cm reversed-phase column (specifications: PePSepC18, 1.9 μm, 75 μm×25 cm, manufacturer: Bruker, country of origin: Germany). The mobile phase was an acetonitrile-water-formic acid system, specifically mobile phase A (0.1% formic acid aqueous solution) and mobile phase B (0.1% formic acid acetonitrile solution). The chromatographic separation procedure was as follows: the column was first equilibrated with 100% mobile phase A, and then the sample was directly loaded onto the column using an autosampler. Gradient separation was then performed, with the flow rate maintained at 300 nL / min for a total gradient duration of 60 min. The proportion of mobile phase B changed as follows: initially maintained at 2% for 0 min, then gradually increased to 22% over 45 min, followed by 37% over 5 min, then 80% over another 5 min, and finally maintained at 80% for 5 min. For mass spectrometry data acquisition, the mass spectrometer was used in DDA mode with a scan range set to 100–1700 m / z. During PASEF MS / MS scanning, the collision energy showed a linear increasing trend with ion mobility, specifically increasing from 20 eV (corresponding to 1 / K0 = 0.6 Vs / cm²) to 59 eV (corresponding to 1 / K0 = 1.6 Vs / cm²).

[0082] Figures 37-59The results shown in Tables 1 and 2 indicate that the evaluation of phosphorylated protein content in the lower semen of the Control group (fresh semen) and the Requimod R848 group showed that the protein bands were normally distributed without significant degradation, the chromatographic baseline was stable, the peaks were uniform and had high overlap, the protein extraction was stable without significant impurity interference, and the enzymatic hydrolysis efficiency reached 91.56%, which can be used for subsequent analysis. A total of 4176 phosphorylated peptides (4154 of which were quantifiable) and 20122 phosphorylated proteins (19935 of which were quantifiable) were identified. Characterization of phosphorylated proteins revealed that differentially phosphorylated peptides were mainly distributed in the range of 7–38 amino acid residues (with the highest proportion in the 10–20 residue range). Proteins containing one and two phosphorylation sites numbered 149 and 205, respectively. 691 upregulated differentially phosphorylated proteins corresponded to 1036 upregulated peptides, while 314 downregulated differentially phosphorylated proteins corresponded to 410 downregulated peptides. Serine phosphorylation was the predominant differentially phosphorylation site (81.13%), followed by threonine and tyrosine phosphorylation at 16.31% and 2.56%, respectively. Pearson correlation, principal component analysis, and hierarchical clustering analysis confirmed good repeatability and concentrated correlation coefficients within groups, as well as significant separation and differences between groups. Furthermore, 1036 proteins with significantly upregulated phosphorylation levels (such as PI4K2A and PI4K2B) and 410 proteins with significantly downregulated phosphorylation levels were identified, demonstrating high data reliability. Functional enrichment analysis revealed that differentially expressed proteins in the GO analysis were involved in sperm motility-related pathways such as sperm flagellar activity and ciliary assembly (e.g., 19 proteins involved in sperm motility), derived from structures such as motile cilia and sperm flagella in cellular composition, and involved in purine nucleotide binding in molecular function. KEGG analysis enriched 30 significant pathways, including mitophagy and motor protein transduction, involving key proteins such as PI4K2A and GSK3B. COG analysis showed the largest number of signal transduction mechanism proteins (118), while differentially expressed proteins were involved in post-translational modifications and cytoskeleton functions. Motif analysis enriched characteristic motifs such as AKT / PKA ([R..S]) and CK2 ([...ST..]), suggesting that these kinases may regulate sperm motility. Domain prediction revealed core domains directly related to sperm motility, such as the AAA+ATPase domain and protein kinase, ATP binding site, which are involved in processes such as ATP metabolism and flagellar assembly. Subcellular localization showed that 46.61% of differentially phosphorylated proteins were located in the cytoplasm, 30.59% in the nucleus, and the remainder were distributed in extracellular secretory proteins, mitochondria, and other sites.PPI network analysis revealed that phosphorylated differentially expressed proteins form a complex interaction network, with key node proteins potentially playing a central role in signal transduction. Further analysis of key differentially expressed proteins (TLR7, TLR8, PI4K2A, etc.) showed that TLR7 / 8 may indirectly regulate PI4K2A, PI4K2B, and PIP5K1A through CD4 and PTEN. Molecular docking showed that TLR8 and PI4K2A bind stably to their ligands, while TLR7 binds weakly to CD4.

[0083] Table 1. Statistics of peptide cleavage sites

[0084] Table 2. Key Differences and Key Protein Molecular Docking Sites

[0085] Example 14

[0086] Validation of key targets in the goat sperm TLR7 / 8 signaling pathway

[0087] (1) The control group (fresh semen lower layer) and the sorted X semen (lower layer) from the R848 group were transferred to centrifuge tubes and centrifuged at 1200 g for 5 min. The supernatant was discarded and the precipitate was retained. The precipitate was washed three times with PBS (1200 g, 5 min). After removing the supernatant, cell lysis buffer (RIPA:PMSF=100:1) was added according to the cell volume. After mixing, the precipitate was placed on ice for 30 min. During this period, the precipitate was pipetted once every 5 min to promote cell lysis. Finally, the semen samples were centrifuged at 4 ℃ (12000 g, 15 min). The supernatant was used to determine the total protein concentration using the BCA method. (2) Add 10% SDS and protein loading buffer (5×) to the supernatant at a ratio of 4:1, boil for 10 min to denature the protein, cool and store in a -80℃ freezer. (3) Using FuturePAGE protein precast gel, add the denatured protein into the gel wells (30 μg / well) and electrophoresis at 100 V for 90 min; (4) Transfer the target protein onto a PVDF membrane and perform the transfer at 100 V for 60 min; place the PVDF membrane in 5% skim milk blocking solution and block at room temperature for 3 h; (5) Place the PVDF membrane in the corresponding primary antibody solution and incubate overnight at 4°C; then wash the PVDF membrane 3 times with TBST (10 min / time); then immerse the PVDF membrane in the corresponding secondary antibody and incubate at 37°C for 2 h; (6) Take out the PVDF membrane and place it in the box. Wash it with TBST 6 times (5 min / time); prepare ECL luminescent solution at a ratio of 1:1 for later use. (7) The PVDF membrane was developed using a chemiluminescence analyzer, and the protein expression level was analyzed using ImageJ software.

[0088] Figures 60-62 The results shown in Table 3 indicate that, compared with the control group, the expression of PI4K2A, PI4K2B, PIP5K1A, and PTEN proteins in the lower sperm layer of the requimide R848 group was significantly downregulated, while the expression of CD4, p-PTEN, GSKα / β, and p-GSKα / β proteins was significantly upregulated. There was no significant difference in AKT expression between p-AKT and requimide R848. Furthermore, the phosphorylation levels of GSKα / β and PTEN in the sperm of the R848 treatment group were significantly increased, while AKT phosphorylation showed no significant change. Immunofluorescence staining analysis revealed the localization of key differentially expressed proteins. In the control group, PI4K2A was highly fluorescently expressed in the sperm head, while its fluorescence intensity was significantly reduced and some sperm did not express it after treatment with retimidoc R848. In the control group, PI4K2B was expressed in the sperm head and mid and main segments of the tail. After treatment with retimidoc R848, its localization shifted to the equator and neck junction of the sperm head, and some sperm showed low expression in the acrosome. In the control group, PIP5K1A was mainly expressed in the sperm head (weakly expressed in the main segment of the tail), while after treatment with retimidoc R848, it was only weakly expressed in the sperm head. Further flow cytometry analysis showed that the expression levels of PI4K2A (45.53%±9.88%) and PI4K2B (71.20±1.82%) in the requimomod R848 group were significantly lower than those in the control group (90.67%±3.27%), while the expression level of PIP5K1A was not significantly different from that in the control group. This suggests that the requimomod R848 does not act on the specific target of X sperm.

[0089] Table 3. Flow Cytometry Analysis of Phosphorylated Differential Proteins

[0090] As demonstrated by the above embodiments, this invention utilizes non-targeted metabolomics and targeted amino acid metabolomics technologies to comprehensively analyze the metabolite profiles of goat X sperm before and after R848 treatment. The results show that R848 treatment significantly altered 380 metabolites in sperm, including key metabolites such as adenine, L-methionine, glutathione, and inositol, and significantly enriched them in 27 metabolic pathways, including glycine / serine / threonine metabolism, the tricarboxylic acid cycle, and purine metabolism. This indicates that R848 affects the motility of X sperm by interfering with energy metabolism and the performance of the antioxidant system. Further phosphorylated proteomics analysis identified 1036 proteins with significantly upregulated phosphorylation levels and 410 proteins with downregulated levels. Key signaling nodes, such as PI4K2A, PI4K2B, and PTEN, showed significant changes in expression and phosphorylation status, suggesting that these proteins could serve as novel targets for the activation of the TLR7 / 8 signaling pathway and its influence on downstream phosphorylation networks.

[0091] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A goat X and Y sperm sorting solution, characterized in that, The components using water as a solvent include the following concentrations: Glucose 24–27 g / L, citric acid 15–18 g / L, sodium EDTA 14–17 g / L, sodium chloride 55–59 g / L, sodium bicarbonate 19–22 g / L, potassium chloride 32–35 g / L, Tris 5.6–6.0 g / L, BSA 0.4–0.8 g / L, and requimomod R848 1.0 μmol / L.

2. The application of the goat X and Y sperm sorting solution according to claim 1 in the preparation of a reagent for efficient sorting of goat X and Y sperm.

3. A goat X and Y sperm sorting reagent, characterized in that, Includes goat X and Y sperm sorting solution, washing solution, and requimod R848; The goat X and Y sperm sorting solution is the goat X and Y sperm sorting solution as described in claim 1; The washing solution, with water as the solvent, comprises the following components at the following concentrations: Tris 180~220 mM / 100mL, citric acid 70~90 mM / 100mL, and fructose 60~70 mM / 100mL.

4. The application of the goat X and Y sperm sorting reagent according to claim 3 in the efficient sorting of goat X and Y sperm.

5. A method for separating X and Y sperm from goats, characterized in that, Includes the following steps: (1) The components are prepared according to the goat X and Y sperm sorting reagent as described in claim 3; (2) Mix fresh goat semen with goat X and Y sperm separation solution, centrifuge, discard the supernatant, and obtain semen precipitate; (3) Mix the semen precipitate with the goat X and Y sperm sorting solution and adjust the sperm density to 5~7×10⁻⁶. 8 cells / mL, to obtain mixed semen; (4) Add Requimod R848 to the mixed semen at a final concentration of 0.7~0.9 μmol / L, incubate for 18~22 min, discard the upper layer of semen, take the lower layer of semen and mix it with the goat X and Y sperm sorting solution, add Requimod R848 at a final concentration of 0.5~0.7 μmol / L, incubate for 8~12 min, discard the upper layer of semen, take the lower layer of semen and mix it with the goat X and Y sperm sorting solution, centrifuge, discard the supernatant, and take the precipitate; resuspend the precipitate with washing solution to obtain goat X sperm; Add 0.3–0.5 μmol / L of Requimod R848 to the mixed semen and incubate for 28–32 min. Collect the supernatant. Mix the supernatant with goat X and Y sperm sorting solution, add 0.1–0.3 μmol / L of Requimod R848, and incubate for 9–11 min. Collect the supernatant and mix with the goat X and Y sperm sorting solution, centrifuge, discard the supernatant, and collect the precipitate. Resuspend the precipitate in washing solution to obtain goat Y sperm. In step (2), the centrifugation speed is 600×g~800×g, and the centrifugation time is 4~6 min; In step (4), the centrifugation speed is 700×g~900×g and the centrifugation time is 4~6 min.

6. The application of the method of claim 5 in the efficient sorting of X and Y sperm from goats.

7. Application of Requimod R848 as a sorting agent for goat X and Y sperm in inhibiting normal motility of goat X sperm.

8. Application of Requimod R848 as a sorting reagent for goat X and Y sperm in interfering with the normal motility of goat X sperm.

9. The application according to claim 7 or 8, characterized in that, Requimod R848 inhibits or interferes with normal motility of goat X sperm by downregulating the expression levels of PI4K2A and PI4K2B proteins in goat X sperm and altering their subcellular localization, while increasing the phosphorylation level of PTEN protein.

10. Application of PI4K2A protein expression level, PI4K2B protein expression level, or PTEN protein phosphorylation level as targets in the preparation of reagents to improve abnormal motility of goat X sperm; The abnormal movement of goat X sperm was caused by the separation of goat X and Y sperm by requitomod R848 as a sorting reagent.

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