Biotoxicity detection method for quail feed containing microplastics

Abstract

The invention provides a biotoxicity detection method for quail feed containing microplastics, and relates to the technical field of biotoxicity detection. According to the biotoxicity detection method of the micro-plastic-containing quail feed, a multi-factor micro-plastic exposure model is constructed, histomorphology, biochemical index detection, molecular biology and omics analysis technologies are combined, and the physiological toxicity of micro-plastic exposed through the feed to the intestinal tract, liver and immune system of quails is systematically detected; the comprehensive and accurate evaluation on the toxicity of the micro-plastic-containing quail feed is realized. According to the method, the detection dimension covers the organ form, biochemical metabolism, flora balance and molecular mechanism, the operation process is standardized, the detection result is high in reliability, and technical support can be provided for microplastic pollution risk management and control and food safety evaluation in livestock and poultry feed.

Description

Technical Field

[0001] This invention relates to the field of biotoxicity detection technology, specifically a method for detecting the biotoxicity of quail feed containing microplastics. Background Technology

[0002] Microplastics, a new type of pollutant widely present in the environment, have a diameter of less than 5 mm and can enter agricultural ecosystems through wastewater irrigation, plastic packaging, and atmospheric deposition, thus accumulating in crops and livestock feed. Quail, as a commonly used model organism, is characterized by its short growth cycle, high metabolism, and sensitivity to pollutants. If microplastics are present in their feed, they may damage the physiological functions of quail through the food chain and even indirectly affect human health.

[0003] Current methods for detecting microplastic toxicity mostly focus on aquatic organisms or single organs. Methods for detecting the biotoxicity of microplastics in livestock and poultry feed are still incomplete, suffering from limitations such as single detection dimensions, incomplete indicator coverage, and a lack of systematic assessment frameworks. Existing methods often only detect the morphological effects of a single microplastic concentration on a particular organ, without considering comprehensive analyses of gut microbiota balance, molecular mechanisms, and immunotoxicity. These methods fail to fully reflect the toxic effects of microplastics on livestock and poultry through feed exposure and cannot meet the practical needs of feed safety management and environmental risk assessment.

[0004] Therefore, establishing a multi-dimensional and systematic method for detecting the biotoxicity of microplastic-containing quail feed is of great significance for ensuring the safety of livestock and poultry farming and food safety. Summary of the Invention

[0005] (a) Technical problems to be solved To address the shortcomings of existing technologies, this invention provides a method for detecting the biotoxicity of quail feed containing microplastics. By constructing a standardized microplastic exposure model and combining it with multidisciplinary detection technologies, a comprehensive and accurate assessment of the toxicity of microplastic feed can be achieved, solving the problems of single-dimensionality and low reliability of existing detection methods.

[0006] (II) Technical Solution To achieve the above objectives, the present invention provides the following technical solution: a method for detecting the biotoxicity of quail feed containing microplastics, comprising the following steps: Step 1: Construct a microplastic exposure model: One-day-old Japanese quail were selected as the research subjects, and at least one of polypropylene, polyethylene, and polystyrene was selected as the target microplastics. The quail were randomly divided into a control group and an experimental group. The control group was fed a basic feed, while the experimental group was fed feed with different concentrations of microplastics added, namely 1 mg / kg, 10 mg / kg, and 100 mg / kg. The feed was changed daily and thoroughly mixed for 20 minutes. After 8 weeks of continuous exposure, the quail were sacrificed, and intestinal and liver tissues were collected, fixed with a fixative, and frozen at -80℃. Step 2: Detect the physiological toxicity of microplastics to the quail's intestines: After processing frozen intestinal tissue, the structure of intestinal villi and changes in the mucus layer were observed under a microscope. The contents of triglycerides (TG), alanine aminotransferase (ALT), aspartate aminotransferase (AST), diamine oxidase (DAO), glutathione (GSH), catalase (CAT), total superoxide dismutase (T-SOD), and total antioxidant capacity (T-AOC) in the intestinal tissue were detected using a kit. The changes in Shannon index, Chao1 index, and OTU in the intestinal contents were analyzed using PCR combined with gel electrophoresis, fluorescence detection, and sequencing technology to characterize the balance and stability of the intestinal flora. Step 3: Detect the physiological toxicity of microplastics to quail liver: After processing frozen liver tissue, the fat deposition in the liver tissue was observed under a microscope. RNA was extracted from the liver tissue using a nucleic acid extractor, and the liver transcriptome characteristics were analyzed by sequencing. Qualitative and quantitative analysis of the liver tissue was performed using LC-MS to study changes in liver metabolomics. The levels of tumor necrosis factor (TNF-α) and interleukin-1β (IL-1β) in the liver tissue were detected by enzyme-linked immunosorbent assay (ELISA), and the expression level of peroxisome proliferator-activated receptor (PPAR) was detected by real-time quantitative PCR. The levels of triglycerides (TG) and total cholesterol (TCH) in the liver were detected by an automated biochemical analyzer, and the changes in monosaccharides and organic acids in the liver were analyzed by high-performance liquid chromatography (HPLC). Step 4: Test the toxicity of microplastics to the quail's immune system: Total proteins from liver tissue and cells were extracted using RIPA lysis buffer containing PMSF, separated by gel electrophoresis, and transferred to PVDF membranes. The membranes were then incubated with HRP-conjugated secondary antibody, and protein expression was analyzed using a chemiluminescence imaging system. After thawing and centrifugation, liver tissue was seeded into 96-well plates and cultured in medium containing different concentrations of microplastics. The viability of liver cancer cells was detected using a CCK8 assay. The expression level of chemokine receptor genes in tissue extracts was detected using real-time quantitative PCR. Mononuclear cells were induced into macrophages, and different concentrations of microplastics were added. After washing with PBS and incubation with CCK8 reagent for 2 hours, cell viability was detected using a microplate reader. Macrophages were stained with fluorescence, and changes in mitochondrial levels and mitochondrial membrane potential were observed using a fluorescence microscope. Step 5: Toxicity Assessment Based on the above test results, the biotoxicity level of quail feed containing microplastics was determined from multiple dimensions, including organ morphological damage, biochemical metabolic disorders, gut microbiota imbalance, abnormal immune function, and changes in molecular expression.

[0007] Preferably, in step one, the fixative is a 4% paraformaldehyde solution, and the tissue fixation time is 24-48 hours.

[0008] Preferably, in step two, the intestinal tissue processing includes gradient dehydration, paraffin embedding, sectioning, and HE staining, and the section thickness is 5-8 μm.

[0009] Preferably, in step three, the RNA extraction is performed using the Trizol method, the sequencing depth of the transcriptome sequencing is not less than 10G, and the metabolomics analysis is performed using ultra-high performance liquid chromatography-tandem mass spectrometry.

[0010] Preferably, in step four, the gel electrophoresis uses a 10% SDS-PAGE gel, and the electrophoresis voltage is 80-120V, the transfer current is 300mA, and the transfer time is 90-120min.

[0011] Preferably, in step four, the inducer for converting monocytes into macrophages is macrophage colony-stimulating factor (M-CSF), and the induction time is 7-10 days.

[0012] Preferably, in step one, each group of quails has 3 parallel replicates, each replicate contains 10-15 quails, and the rearing environment temperature is controlled at 25-28℃, relative humidity at 55-65%, and the light cycle is 12h light / 12h dark.

[0013] Preferably, in step two, the gut microbiota analysis uses a high-throughput sequencer to sequence the V3-V4 region of the 16S rRNA gene.

[0014] Preferably, in step three, when analyzing monosaccharides and organic acids by high performance liquid chromatography, the mobile phase is acetonitrile-0.1% formic acid aqueous solution, and the detection wavelength is 210-254 nm.

[0015] (III) Beneficial Effects This invention provides a method for detecting the biotoxicity of quail feed containing microplastics, which has the following beneficial effects: 1. This invention constructs a multi-factor microplastic exposure model, covering common microplastic types and gradient concentrations, simulating microplastic pollution scenarios in actual feed, and the detection results are closer to the actual situation, providing reliable model support for feed safety assessment.

[0016] 2. Comprehensive detection dimensions, covering the three core organs of the intestine, liver and immune system. It conducts detection from multiple levels such as tissue morphology, biochemical metabolism, microbial balance, molecular expression and cell function, which can systematically reveal the toxic effects and mechanisms of action of microplastics, overcoming the shortcomings of existing methods with only one dimension. Attached Figure Description

[0017] Figure 1 This is a schematic diagram of the biotoxicity detection method of the present invention. Detailed Implementation

[0018] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0019] Example 1: like Figure 1 As shown in the figure, this invention provides a method for detecting the biotoxicity of quail feed containing microplastics, comprising five steps: microplastic exposure model construction, intestinal toxicity detection, liver toxicity detection, immune system toxicity detection, and comprehensive toxicity assessment, as detailed below: Step 1: Constructing a microplastic exposure model Healthy one-day-old Japanese quails were selected as research subjects and randomly divided into groups to ensure that there were no significant differences in initial weight and health status among the groups. Three common environmental microplastics—polypropylene, polyethylene, and polystyrene—were selected as target pollutants. A control group (no microplastics added) and three experimental groups (microplastic concentrations of 1 mg / kg, 10 mg / kg, and 100 mg / kg, respectively) were set up, with three replicates per group and 10-15 quails per replicate.

[0020] Fresh microplastic-containing feed was provided daily. Before replacement, the microplastics were thoroughly mixed with the base feed for 20 minutes to ensure even dispersion. The rearing environment was controlled at a temperature of 25-28℃, relative humidity of 55-65%, and a 12-hour light / 12-hour dark cycle, with free access to food and water. After 8 weeks of continuous exposure, the quails were euthanized by cervical dislocation. The intestinal and liver tissues were quickly separated, surface impurities were removed, and the tissues were fixed in a 4% paraformaldehyde solution for 24-48 hours. Subsequently, they were transferred to an ultra-low temperature freezer at -80℃ for cryopreservation and subsequent testing.

[0021] Step 2: Detecting the physiological toxicity of microplastics to the quail's intestines 2.1 Morphological observation of intestinal tissue The fixed intestinal tissue was removed, dehydrated in a gradient manner (70%, 80%, 90%, 95%, 100% ethanol), cleared with xylene, embedded in paraffin, and then cut into 5-8 μm thick sections for HE staining. The stained sections were observed under an optical microscope, focusing on the height, width, and regularity of the intestinal villi, as well as the thickness of the mucus layer. The intestinal villi damage rate and the degree of mucus layer reduction were statistically analyzed to characterize the extent of intestinal barrier function impairment.

[0022] 2.2 Detection of intestinal biochemical indicators Frozen intestinal tissue was collected, and the levels of triglycerides (TG), alanine aminotransferase (ALT), aspartate aminotransferase (AST), diamine oxidase (DAO), glutathione (GSH), catalase (CAT), total superoxide dismutase (T-SOD), and total antioxidant capacity (T-AOC) were measured according to the kit instructions. DAO reflects intestinal mucosal integrity, GSH, CAT, T-SOD, and T-AOC characterize intestinal antioxidant capacity, and TG, ALT, and AST reflect the degree of intestinal metabolic disorder.

[0023] 2.3. Intestinal flora analysis Intestinal contents were collected, and total bacterial DNA was extracted aseptically. The V3-V4 region of the 16S rRNA gene was amplified by PCR. After verifying the purity of the amplified products by gel electrophoresis, high-throughput sequencing was performed. After quality control filtering of the sequencing data, the changes in the Shannon index (reflecting bacterial diversity), Chao1 index (reflecting bacterial richness), and OTU (operational taxa) were analyzed. The relative abundance of beneficial bacteria (such as lactobacilli and bifidobacteria) and harmful bacteria (such as Escherichia coli and Salmonella) was statistically analyzed to clarify the mechanism by which microplastics affect the balance and stability of the intestinal flora.

[0024] Step 3: Detect the physiological toxicity of microplastics to quail livers. 3.1. Observation of liver histomorphology After fixation, liver tissue was dehydrated, embedded, sectioned, and stained with Oil Red O. It was then observed under an optical microscope to observe the number, size, and distribution of fat droplets in the liver tissue, count the area of ​​fatty degeneration, and characterize the damage of microplastics to liver lipid metabolism.

[0025] 3.2 Liver transcriptome analysis Frozen liver tissue was harvested, and total RNA was extracted using the Trizol method. After purification using a nucleic acid extractor, RNA purity and integrity were assessed (OD260 / OD280 ratio of 1.8-2.0). Qualified RNA samples were sent to a sequencing platform for transcriptome sequencing (sequencing depth not less than 10G). Differential gene expression analysis was used to screen for differentially expressed genes related to liver inflammation and metabolism, clarifying the impact of microplastics on liver transcription levels.

[0026] 3.3 Liver metabolomics analysis Liver tissue was homogenized and centrifuged to obtain the supernatant. Qualitative and quantitative analysis was performed using ultra-high performance liquid chromatography-tandem mass spectrometry (LC-MS). Through metabolite identification and differential metabolite screening, the changes in triglyceride (TG), total cholesterol (TCH), monosaccharides, and organic acids were analyzed to reveal the impact of microplastics on liver metabolic function.

[0027] 3.4 Detection of liver inflammation and related factors Enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in liver tissue. Both factors are important inflammatory markers, and elevated levels indicate an inflammatory response in the liver. Real-time quantitative PCR (qPCR) was used to detect the expression level of peroxisome proliferator-activated receptor (PPAR). PPAR is involved in lipid metabolism and inflammation regulation, and changes in its expression can reflect the metabolic and inflammatory status of the liver. Furthermore, an automated biochemical analyzer was used to detect the levels of triglycerides (TG) and total cholesterol (TCH) in the liver, and high-performance liquid chromatography (HPLC) was used to analyze the types and contents of monosaccharides and organic acids to further verify the presence of hepatic metabolic disorders.

[0028] Step 4: Test the toxicity of microplastics to the quail's immune system 4.1 Liver tissue protein analysis Total protein was extracted from liver tissue and cells using RIPA lysis buffer containing PMSF. Protein concentration was determined using the BCA method. After adjusting the protein concentration across groups to be consistent, loading buffer was added and the samples were boiled for denaturation. The denatured protein samples were then transferred to a 10% SDS-PAGE gel for electrophoresis (stacking gel voltage 80V, separating gel voltage 120V). After electrophoresis, the proteins were transferred to a PVDF membrane (transfer current 300mA, transfer time 90-120min). The PVDF membrane was blocked with 5% skim milk for 1 hour, incubated overnight with primary antibody, washed with TBST, and then incubated with HRP-conjugated secondary antibody for 1 hour. After washing again, protein bands were detected using a chemiluminescence imaging system to analyze the expression levels of immune-related proteins.

[0029] 4.2 Cell viability assay After thawing frozen liver tissue, it was rinsed with sterile saline, minced, digested with trypsin, and centrifuged to collect hepatocytes. The hepatocytes were then seeded into 96-well plates and cultured for 24 hours with culture medium containing different concentrations of microplastics. 10 μL of CCK8 reagent was added to each well, and incubation continued for 2 hours. The absorbance at 450 nm was then measured using a microplate reader to calculate cell viability and determine the effect of microplastics on hepatocyte viability and whether they induced the formation of liver cancer cells.

[0030] Meanwhile, quail peripheral blood mononuclear cells were isolated and induced with macrophage colony-stimulating factor (M-CSF) for 7-10 days to obtain mature macrophages. Macrophages were seeded in 96-well plates, cultured with different concentrations of microplastics for 24 hours, washed three times with PBS, and then incubated with CCK8 reagent for 2 hours. The absorbance was then measured using a microplate reader to analyze the effect of microplastics on macrophage viability.

[0031] 4.3 Gene Expression Detection The expression level of chemokine receptor genes in liver tissue extracts was detected by real-time quantitative PCR (qPCR). Chemokine receptors are involved in the migration and activation of immune cells, and changes in their expression can reflect the functional status of the immune system.

[0032] 4.4 Macrophage Function Detection Induced mature macrophages were seeded in culture dishes, and after culturing for 24 hours with different concentrations of microplastics, they were washed three times with PBS to remove unphagocytosed microplastics. Macrophages were then stained with fluorescent dyes (JC-1 staining solution for mitochondria and DiI staining solution for cell membranes) and observed under a fluorescence microscope. The number, morphology, and mitochondrial membrane potential changes of macrophage mitochondria were recorded. Mitochondrial dysfunction can directly affect the phagocytic and immune activation capabilities of macrophages, thus reflecting the extent of damage to the immune system.

[0033] Step 5: Toxicity Assessment Based on the above test results for the gut, liver, and immune system, a multi-dimensional toxicity assessment system was established, classifying the biotoxicity of microplastic-containing quail feed into four levels: Non-toxic: All test indicators (tissue morphology, biochemical indicators, microbial index, gene expression, etc.) showed no significant difference from the control group (P≥0.05), and the quail's physiological functions were normal; Low toxicity: Significant changes were observed in 1-2 test indicators (P<0.05), but there was no obvious organ morphological damage, and the physiological functions of the quail were basically normal; Toxicity: 3-5 test indicators are significantly abnormal (P<0.05), mild organ morphological damage occurs (such as slight breakage of intestinal villi, small amount of fat droplet deposition in the liver), and the immune system function is slightly decreased; High toxicity: ≥6 test indicators were significantly disordered (P<0.05), accompanied by obvious organ damage (such as severe intestinal villi breakage and large-area fatty degeneration of the liver) and immunosuppression (such as significantly reduced macrophage activity and downregulated expression of immune proteins), and the growth and development of quail were significantly inhibited.

[0034] Example 2: Based on Example 1, the following detailed description is provided: a method for detecting the biotoxicity of quail feed containing microplastics, with the following specific steps: Step 1: Constructing a microplastic exposure model One hundred and twenty healthy one-day-old Japanese quails were randomly divided into four groups, with three replicates per group and ten quails per replicate. The control group was fed a basal diet, while the experimental groups were fed diets supplemented with 1 mg / kg polypropylene, 10 mg / kg polypropylene, and 100 mg / kg polypropylene, respectively. Fresh feed was provided daily, mixed for 20 minutes before feeding. The rearing environment was maintained at 26°C, 60% relative humidity, and a 12-hour light / 12-hour dark cycle. After eight weeks of continuous exposure, the quails were euthanized, and intestinal and liver tissues were collected, fixed with 4% paraformaldehyde for 24 hours, and then frozen at -80°C.

[0035] Step 2: Detecting the physiological toxicity of microplastics to the quail's intestines After dehydration, embedding, sectioning (5 μm), and HE staining, microscopic observation of intestinal tissue showed that the height of intestinal villi in the 100 mg / kg experimental group was significantly reduced (P < 0.05), and the thickness of the mucus layer was reduced by 30%. Kit detection showed that the DAO content in the 100 mg / kg experimental group increased by 25%, and the T-SOD activity decreased by 20%. Microbial community analysis showed that the Shannon index in the 100 mg / kg experimental group decreased by 0.3, the abundance of beneficial lactic acid bacteria decreased by 15%, and the abundance of harmful Escherichia coli increased by 20%.

[0036] Step 3: Detect the physiological toxicity of microplastics to quail livers. Oil Red O staining of liver tissue showed that the fatty degeneration area in the 100 mg / kg experimental group reached 15%; transcriptome sequencing screened 87 differentially expressed genes, including 12 inflammation-related genes; LC-MS detection showed that the TG content in the 100 mg / kg experimental group increased by 30% and the lactate content increased by 25%; ELISA detection showed that the TNF-α and IL-1β levels in the 100 mg / kg experimental group increased by 40% and 35%, respectively; qPCR detection showed that the PPAR expression level in the 100 mg / kg experimental group decreased by 25%.

[0037] Step 4: Test the toxicity of microplastics to the quail's immune system Protein analysis showed that immunoglobulin G expression decreased by 20% in the 100 mg / kg experimental group; CCK8 assay showed that hepatocyte survival rate decreased by 15% and macrophage survival rate decreased by 25% in the 100 mg / kg experimental group; qPCR assay showed that chemokine receptor gene expression decreased by 30% in the 100 mg / kg experimental group; fluorescence microscopy showed that the number of mitochondria in macrophages decreased by 20% and mitochondrial membrane potential decreased by 15% in the 100 mg / kg experimental group.

[0038] Step 5: Toxicity Assessment Based on the comprehensive test results, the 100 mg / kg polypropylene experimental group showed significant abnormalities in 7 test indicators, as well as obvious organ damage and immune function suppression, and was therefore determined to be highly toxic at this concentration of microplastic-containing feed; the 10 mg / kg experimental group showed significant abnormalities in 3 test indicators and mild organ damage, and was therefore determined to be moderately toxic; the 1 mg / kg experimental group showed a significant change in 1 test indicator and no organ damage, and was therefore determined to be low toxic; the control group showed no significant abnormalities and was therefore determined to be non-toxic.

[0039] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for detecting the biotoxicity of quail feed containing microplastics, characterized in that: Includes the following steps: Step 1: Construct a microplastic exposure model: One-day-old Japanese quail were selected as the research subject, and at least one of polypropylene, polyethylene, and polystyrene was selected as the target microplastic. Quails were randomly divided into a control group and an experimental group. The control group was fed a basic feed, while the experimental group was fed feed with different concentrations of microplastics added, namely 1 mg / kg, 10 mg / kg, and 100 mg / kg. Fresh feed was added daily and the feed was thoroughly mixed for 20 minutes. After 8 weeks of continuous exposure, the quails were euthanized, and intestinal and liver tissues were collected, fixed with a fixative, and then frozen and stored at -80℃. Step 2: Detect the physiological toxicity of microplastics to the quail's intestines: After processing frozen intestinal tissue, the structure of intestinal villi and changes in the mucus layer were observed under a microscope. The contents of triglycerides (TG), alanine aminotransferase (ALT), aspartate aminotransferase (AST), diamine oxidase (DAO), glutathione (GSH), catalase (CAT), total superoxide dismutase (T-SOD), and total antioxidant capacity (T-AOC) in the intestinal tissue were detected using a kit. The changes in Shannon index, Chao1 index, and OTU in the intestinal contents were analyzed using PCR combined with gel electrophoresis, fluorescence detection, and sequencing technology to characterize the balance and stability of the intestinal flora. Step 3: Detect the physiological toxicity of microplastics to quail liver: After processing the frozen liver tissue, the fat deposition in the liver tissue was observed under a microscope; RNA was extracted from the liver tissue using a nucleic acid extractor, and the liver transcriptome characteristics were analyzed by sequencing. LC-MS was used to perform qualitative and quantitative analysis of liver tissue to study changes in liver metabolomics; The levels of tumor necrosis factor (TNF-α) and interleukin-1β (IL-1β) in liver tissue were detected by enzyme-linked immunosorbent assay (ELISA), and the expression level of peroxisome proliferator-activated receptor (PPAR) was detected by real-time quantitative PCR. The levels of triglycerides (TG) and total cholesterol (TCH) in the liver were detected by an automated biochemical analyzer, and the changes in monosaccharides and organic acids in the liver were analyzed by high performance liquid chromatography (HPLC). Step 4: Test the toxicity of microplastics to the quail's immune system: Total proteins from liver tissue and cells were extracted using RIPA lysis buffer containing PMSF, separated by gel electrophoresis, transferred to PVDF membranes, and incubated with HRP-conjugated secondary antibody. Protein expression was analyzed using a chemiluminescence imaging system. After thawing and centrifugation, liver tissue was seeded into 96-well plates and cultured in microplastic medium of different concentrations. The viability of liver cancer cells was detected using a CCK8 assay kit. The expression level of chemokine receptor gene in tissue extracts was detected by real-time quantitative PCR; monocytes were induced into macrophages and then microplastics of different concentrations were added. After washing with PBS and incubation with CCK8 reagent for 2 h, cell viability was detected by microplate reader; macrophages were stained with fluorescence and changes in mitochondrial level and mitochondrial membrane potential were observed by fluorescence microscopy. Step 5: Toxicity Assessment Based on the above test results, the biotoxicity level of quail feed containing microplastics was determined from multiple dimensions, including organ morphological damage, biochemical metabolic disorders, gut microbiota imbalance, abnormal immune function, and changes in molecular expression.

2. The method for detecting the biotoxicity of microplastic-containing quail feed according to claim 1, characterized in that: In step one, the fixative is a 4% paraformaldehyde solution, and the tissue fixation time is 24-48 hours.

3. The method for detecting the biotoxicity of microplastic-containing quail feed according to claim 1, characterized in that: In step two, the intestinal tissue processing includes gradient dehydration, paraffin embedding, sectioning, and HE staining, with the section thickness being 5-8 μm.

4. The method for detecting the biotoxicity of microplastic-containing quail feed according to claim 1, characterized in that: In step three, the RNA extraction is performed using the Trizol method, the transcriptome sequencing depth is no less than 10G, and the metabolomics analysis is performed using ultra-high performance liquid chromatography-tandem mass spectrometry.

5. The method for detecting the biotoxicity of microplastic-containing quail feed according to claim 1, characterized in that: In step four, the gel electrophoresis uses 10% SDS-PAGE gel, with an electrophoresis voltage of 80-120V, a transfer current of 300mA, and a transfer time of 90-120min.

6. The method for detecting the biotoxicity of microplastic-containing quail feed according to claim 1, characterized in that: In step four, the agent for inducing monocytes into macrophages is macrophage colony-stimulating factor (M-CSF), and the induction time is 7-10 days.

7. The method for detecting the biotoxicity of microplastic-containing quail feed according to claim 1, characterized in that: In step one, each group of quails is set up with 3 parallel replicates, each replicate containing 10-15 quails. The temperature of the rearing environment is controlled at 25-28℃, the relative humidity is 55-65%, and the light cycle is 12h light / 12h dark.

8. The method for detecting the biotoxicity of microplastic-containing quail feed according to claim 1, characterized in that: In step two, the gut microbiota analysis uses a high-throughput sequencer to sequence the V3-V4 region of the 16S rRNA gene.

9. The method for detecting the biotoxicity of microplastic-containing quail feed according to claim 1, characterized in that: In step three, when analyzing monosaccharides and organic acids by high performance liquid chromatography, the mobile phase is acetonitrile-0.1% formic acid aqueous solution, and the detection wavelength is 210-254 nm.

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