Male-specific pathogen-free (SPF) C57BL/6 mice (6−7 weeks old, 18 ± 1 g) were obtained from the Sibeifu Animal Company (SCXK(JING)-2019-0010, Beijing, China) and housed at the Animal Experimental Center of the Shanxi University of TCM. They were placed in a controlled environment (25 ± 2°C, 55 ± 5% humidity, 12-h light/dark cycle) with free access to food and water. They were acclimated for one week prior to the experiment. All animal procedures followed the National Institute of Health (NIH) guidelines and were approved by the Shanxi University of Chinese Medicine Animal Ethics Committee (No. AWE202303381).

Mice were randomly divided into control, model, and acupuncture (ACU) groups with 14 mice per group. This was accomplished using a random number table. The model and ACU groups received intraperitoneal injections of cyclophosphamide (HY-17420, MedChemExpress, NJ, USA) at 50 mg/kg/day to induce an asthenozoospermia model (20), while the control group received an equivalent volume of 0.9% sodium chloride solution that was administered for five consecutive days. The ACU group mice underwent acupuncture treatment for two weeks one day after model induction. Disposable acupuncture needles (0.25 mm×13 mm) were inserted bilaterally at the “Zhibian (BL 54)” points (located in the bony fissure where the line between the greater trochanter and the midpoint of the fourth sacral vertebra meets) and angled toward the “Shuidao (ST 28)” points (approximately 8 mm below the junction of the upper 2/3 and lower 1/3 between the xiphoid process and pubic symphysis and 3 mm lateral to the midline) (16) at a depth of 5 mm for 20 min each session once daily. The control and model group mice remained fixed without further interventions. The mice were anesthetized with isoflurane after the treatments, and samples were collected for subsequent experiments.

A total of 12 mice were randomly divided into two groups (FMT-M and FMT-A). A cyclophosphamide injection (50 mg/kg/d) was first administered intraperitoneally for five consecutive days to induce asthenozoospermia in the mice. Gavage was then used to administer an antibiotic mixture daily for seven days to clear the existing gut microbiota (21). The antibiotic mixture consisted of vancomycin (100 mg/kg), neomycin sulfate (200 mg/kg), metronidazole (200 mg/kg), and ampicillin (200 mg/kg) (MB1260, MB1716, MB2200, and MB1378 were purchased from Meilun Biological, Dalian, China). Model and ACU group fecal samples that were collected in a previous experiment were homogenized in a 0.1 g feces/1 mL phosphate buffer solution (PBS) ratio, and the supernatants were collected using centrifugation at 100 g for 5 min at 4°C. The model and ACU group supernatants were then transferred to the FMT-M and FMT-A mice groups at a dose of 10 mL/kg/d via gavage for seven consecutive days.

All mice in each group were placed into a specialized small animal inhalation anesthesia machine and anesthetized using 2.5% isoflurane. The mice were then placed in a supine position and fixed on a dissection table. The abdominal skin was disinfected with alcohol, and the abdominal skin and muscles were carefully cut along the midline using scissors and forceps to expose the abdominal cavity. The testes and epididymal tissues were gently lifted with forceps and carefully separated from surrounding blood vessels, vas deferens, and other tissues. Excess fat tissue was trimmed off, and the tissues were placed on filter paper to absorb excess moisture. An electronic balance was used to weigh the testes, and the formula for the testicular coefficient (%) was mass of both testes (g)/final body weight of the mouse (g) × 100%.

All mice in each group were anesthetized, and the abdominal skin was disinfected with alcohol. The midline was cut using scissors and forceps to open the abdominal skin and muscle and expose the abdominal cavity. The epididymal tissue was excised to remove any surrounding adipose tissue. The tissues were then cut into small pieces, placed in a petri dish that contained 1 mL of physiological saline, and incubated in a 37°C water bath for 30 min to allow the sperm to be fully released. The supernatant was collected to obtain the epididymal sperm. A total of 20 μL of sperm suspension was placed using a pipette onto a pre-warmed sperm counting chamber at 37°C. This was then analyzed using a computer-assisted sperm analysis (CASA) system (WLJY-9000, Weili Co., Beijing, China). Ten fields of each sample were examined to determine the sperm concentration, viability, and motility (22).

Blood samples were collected using the eyeball removal method. The blood was allowed to stand for 2 h, and it was then centrifuged at 3500 r/min for 15 min at 4°C (centrifuge radius 8.2 cm). The upper serum was collected and stored at –80°C for later analysis. An enzyme-linked immunosorbent assay (ELISA) detection was performed using seven random serum samples from each group. A total of 100 μL of a standard solution was added to each blank and standard well, while 100 μL of the sample was added to each sample well. A pipette was used to thoroughly mix the mixture, and it was incubated at 37°C for 90 min. The liquid was then discarded, and 100 μL of a biotinylated antibody working solution was added to each well for a 1-h incubation at 37°C. The plate was then washed, and the excess liquid was removed (repeated three times). A total of 100 μL of an enzyme conjugate working solution was then added to each well, and the wells were incubated for 30 min at 37°C. The washing step was repeated five times. Next, 90 μL of a substrate solution was added to each well. The plate was then incubated at 37°C in the dark for 15 min. Finally, 50 μL of a stop solution was added to terminate the reaction. A microplate reader (Spectra Max Plus384, Molecular Devices, Sunnyvale, USA) was used to measure the absorbance at 450 nm, and the testosterone (T), follicle-stimulating hormone (FSH), and luteinizing hormone (LH) concentrations were calculated (MU30398, MU30265, MU30382, Bio-swamp, Wuhan, China).

Seven unilateral testicular tissues from each group were randomly selected and fixed in a testicular fixative (G1121, Servicebio, Wuhan, China) for 24 h. The tissues were then transferred to 75% ethanol for 48 h. In addition, seven colon tissues from each group were randomly selected and fixed in a formaldehyde solution for 24 h. Paraffin sections that were 5 μm (RM 2145, LEICA, Wetzlar, Germany) were prepared after dehydration, clearing, paraffin infiltration, and embedding. The paraffin sections were dehydrated using a gradient of 85% and 95% ethanol and then stained with hematoxylin for 3 min. This was followed by eosin staining for 5 min. The sections were dried and mounted with neutral resin and examined under a microscope (ECLIPSE Ci-S, Nikon, Tokyo, Japan) to investigate the testicular and colon tissue histopathological changes. Three different sections were observed for each mouse.

Three epididymal tissue samples were randomly selected from each group and placed in a culture dish. A total of 1 mL of a 0.9% sodium chloride solution was then added. A sterile disposable needle was used to gently puncture the head of the epididymis to release sperm into the culture dish. An appropriate amount of sperm suspension was then evenly pipetted onto a glass slide that was subsequently placed in Dif Quick fixative (G2572, Solarbio, Beijing, China) for 15−20 s. The slide was then stained sequentially in Dif Quick Staining Solution I and II for 5−10 s each. This was followed by 10−15 washes in running water to remove excess dye. After drying, the sperm morphology was observed under a microscope.

The total RNA was extracted from the colon samples of each group and reverse transcribed to synthesize the cDNA. This cDNA was used as a template for PCR amplification under the following conditions: 95°C for 30 s (1 cycle); 95°C for 15 s; and 60°C for 30 s (40 cycles). The Ct values of each well after the reaction were recorded using a PCR machine (CFX 96, Bio-Rad, CA, USA). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control, and the relative expression levels of the target genes were calculated using the 2^–△△CT method. The primer sequences are listed in Table 1 (designed by Servicebio, Wuhan, China).

The total protein was extracted from the testicular and colonic tissues of each mouse group. The bicinchoninic acid assay (BCA) method was used to determine the protein concentrations. Protein samples were loaded into a gel for electrophoresis. This was followed by wet transfer and blocking for 2 h each. The bands were then placed in separate wet chambers, and each chamber was incubated overnight at 4°C with the following antibodies: ZO-1 (A0659, Abclonal, Wuhan, China), Occludin (GB111401, Servicebio, Wuhan, China), Claudin-1 (GB112543, Servicebio, Wuhan, China), Connexin-43 (A11752, Abclonal, Wuhan, China), and N-cadherin (A3045, Abclonal, Wuhan, China). The dilution ratios were 1:1000 for ZO-1, Occludin, Claudin-1, Connexin-43, and N-cadherin antibodies, and 1:3000 for β-actin. The membranes were washed the following day with tris-buffered saline and Polysorbate 20 (TBST) and incubated with the secondary antibody (dilution 1:5000) for 2 h. The membranes were then washed again with TBST, and a chemiluminescent detection solution was added for imaging. The relative expression levels of the target proteins among the different samples were analyzed by comparing the gray values of the bands using Image J software (Bethesda, Maryland, USA).

Seven mice were randomly selected from each group. An iodine tincture was used to disinfect the perianal skin of the mice in the morning following the treatment. The mice were then gently held in a head-up position with their body suspended, and their abdomen was massaged to help them pass fresh feces. Four to five fecal pellets were collected from each mouse and placed into a sterile centrifuge tube. The samples were quickly transferred to liquid nitrogen for 3 h, and these samples were then stored at –80°C for later use. A DNA extraction kit (D5635-02, BioTek, Norcross, GA, USA) was used to extract the microbial DNA from the fecal samples. Amplification and sequencing were performed using gene-specific primers AMV4.5N (5′-AAGCTCGTAGTTGAATTTCG-3′) and AMDGR (5′-CCCAACTATCCCTATT AATCAT-3′). The PCR products were purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA) and quantified using the Quant-iT PicoGreen dsDNA Assay Kit (P7589, Thermo Fisher, Waltham, MA, USA) on a microplate reader (FLx800, BioTek, Norcross, GA, USA). The Illumina NovaSeq platform (NovaSeq 6000, Illumina, San Diego, CA, USA) was used to perform the sequencing with assistance from the Shanghai Personal Biotechnology Co., Ltd. Bioinformatics tools were then used to analyze the raw sequencing data that included the sequence quality control, sequence alignment, operation taxonomic unit (OTU) clustering, and classification steps to determine the microbial species present in the samples and their relative abundances.

Testicular and serum samples were randomly collected from seven mice from each group. Homogenization beads and 500 μL of the extraction solvent (methanol:acetonitrile:water = 2:2:1) were added to the samples. After grinding and centrifugation, the supernatant was transferred to sample vials for analysis using ultra-high performance liquid chromatography with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS) (Vanquish, Thermo Fisher Scientific, Waltham, MA, USA). The chromatographic conditions included an ACQUITY UPLC^® HSS T3 column (150 mm × 2.1 mm, 1.8 μm) with a mobile phase of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B) at a flow rate of 0.35 mL·min^–1, a column temperature of 50°C, and an injection volume of 5 μL. Gradient elution was performed for positive ions (0–1 min, 5% B; 1–24 min, 5%–100% B; 24–28 min, 100% B; 28–30 min, 100%–5% B; and 30–33 min, 5% B) and negative ions (0–1 min, 2% B; 1–18 min, 5%–100% B; 18–22 min, 100% B; 22–25 min, 100%–2% B; and 25–28 min, 2% B). Mass spectrometry conditions included an electrospray ionization (ESI) source with ion source temperatures of 350°C (positive ion mode) and 300°C (negative ion mode), a capillary temperature of 320°C, a sheath gas flow rate of 35 psi, and an auxiliary gas flow rate of 10 psi. The spray voltages were 3.5 kV for positive ions and 2.5 kV for negative ions. The full scan resolution was 70,000 full width at half maximum (FWHM), the secondary scan resolution was 17,500 FWHM, the mass detection range was m/z 100–1,500, the dynamic exclusion duration was 8 s, and the collision energies were 20, 40, and 60 eV. The raw data were then processed and normalized, and the orthogonal partial least squares discriminant analysis (OPLS-DA) model was used to obtain the variable importance for projection (VIP) values > 1 and P values < 0.05 as criteria for the selection of significant differential metabolites (DEMs). DEM screening was conducted in the combined ESI+ and ESI– modes, with overlapping substances between different modes retained based on the larger VIP value. A functional analysis was then performed on the DEMs.

The paraffin-embedded testis tissue sections were first dewaxed to water. The sections were then placed in a retrieval box filled with an ethylenediaminetetraacetic acid (EDTA) antigen retrieval buffer (G1203, Servicebio, Wuhan, China). The sections were then subjected to antigen retrieval in a microwave. The slides were washed in PBS (G0002, Servicebio, Wuhan, China) on a decolorizing shaker for 3×5 min after being naturally cooled. The sections were then slightly dried, and a circle was drawn around the tissue using a histochemical pen. Bovine serum albumin (BSA) (GC305010, Servicebio, Wuhan, China) was added to the circle for a 30 min incubation. The blocking solution was gently removed, and the primary antibodies (Connexin-43 and N-cadherin at 1:200 dilution, Occludin at 1:1000 dilution) were added. The sections were then placed flat in a humidified chamber and incubated overnight at 4°C. After being returned to room temperature the following day, the slides were washed in PBS for 3×5 min. The secondary antibody (GB21303, Servicebio, Wuhan, China) was added and incubated for 50 min at a 1:300 dilution. The sections were then slightly dried, and a 4′,6-diamidino-2-phenylindole (DAPI) staining solution (G1012, Servicebio, Wuhan, China) was added within the circle. This was then incubated in the dark at room temperature for 10 min. The slides were then mounted, and the images were observed under a fluorescence microscope.

GraphPad Prism 7 software was used for the data analysis. Data that followed a normal distribution are presented as means ± standard deviation (SD), and differences between the two groups were analyzed using Student’s t-test. Differences among multiple groups were assessed using a one-way analysis of variance (ANOVA). This was followed by a post-hoc least significant difference (LSD) test. The relationships between the gut microbiota and metabolomics variables were explored using Spearman correlation analyses.

The key indicators for improving symptoms of asthenozoospermia are the testicular mass, the testicular coefficient, and the sperm quality. The model group exhibited significant declines in the testicular mass, the testicular coefficient, the sperm concentration, the sperm motility, and the sperm viability (P < 0.01) compared with the control group. These parameters showed varying degrees of improvement (P < 0.01) after acupuncture treatment ( Figures 1A, B ). Asthenozoospermia is often associated with imbalances in serum hormone levels. In this study, the model group had reduced T, FSH, and LH serum levels compared with the control group (P < 0.01), while the ACU group showed varying degrees of increase in these hormone levels compared with the model group (P < 0.01) ( Figure 1C ). Sperm production primarily occurs in the testis and any structural or functional abnormalities can impact the processes of spermatogenesis and sperm maturation. We observed that the testicular volume in the model group mice decreased (P < 0.01) and had uneven coloration, a roughened surface, and abnormal vascular distribution in some areas ( Figures 1D, E ). H&E staining in the model group revealed disorganized seminiferous tubule structures, deformed or collapsed lumens, reduced or disordered arrangement of spermatogenic cells, and decreased sperm counts ( Figure 1F ). In addition, the interstitial tissue showed signs of widening or edema. Both the testicular volume and pathological morphology of the mice showed varying degrees of improvement after acupuncture treatment. These results indicated that acupuncture effectively enhanced the testicular function and sperm quality in the asthenozoospermic mice.

The gut microbiota is involved in various male reproductive physiological processes, and it can affect sperm quality (23). Therefore, the gut microbiota changes between the different groups were analyzed. The α-diversity index primarily represents richness using the Chao1 index and diversity using the Shannon and Simpson indices. The model group showed significant increases in the Chao1, Shannon, and Simpson indices compared with the control group (P < 0.01, P < 0.05). The ACU group showed significant decreases in the Chao1, Shannon, and Simpson indices compared with the model group (P < 0.01). These results both indicated that the gut microbiota richness and diversity decreased after acupuncture ( Figure 2A ). A principal coordinate analysis (PCoA) and nonmetric multidimensional scaling (NMDS) were used to perform the dimensionality reduction on the multidimensional microbial data to analyze the differences in the β-diversity between samples ( Figure 2B ). The control group and the model group were more dispersedly clustered in the PCoA plot. This result suggested significant changes in the gut microbiota structure between the two groups. The ACU group clustered closer to the control group and further from the model group. This indicated a regulatory effect of acupuncture on the gut microbiota. The NMDS stress value (stress) was 0.126 < 0.2, indicating that the study results were relatively reliable.

Bacteroidota, Firmicutes, Actinobacteriota, and Proteobacteria were the primary dominant phyla of the gut microbiota of each mice group, and they accounted for greater than 90% of the total phyla ( Figure 2C ). The model group showed a significant increase in the relative abundance of Bacteroidota (P < 0.01) and a decrease in the relative abundance of Firmicutes (P < 0.05) compared with the control group, while the differences in Actinobacteriota and Proteobacteria were not significant (P > 0.05). The ACU group showed a significant decrease in the relative abundance of Bacteroidota (P < 0.05) and an increase in the relative abundance of Firmicutes (P < 0.05) compared with the model group. The dominant genera in the gut microbiota of each group were Dubosiella and Paramuribaculum, accounting for greater than 50% of the total genera ( Figure 2D ). However, only Dubosiella and Faecalibaculum showed significant differences between the control group and the model group (P < 0.01, P < 0.05). The relative abundance of Dubosiella increased (P < 0.01), while the relative abundance of Faecalibaculum decreased (P < 0.01) after acupuncture. A Venn diagram was plotted to identify a total of 540 shared species ( Figure 2E ). The Kyoto Encyclopedia of Genes and Genomes (KEGG) database was used to predict the metabolic pathways of the intersecting species. It was revealed that the pathways were primarily related to amino acid biosynthesis, nucleoside, and nucleotide biosynthesis ( Figure 2F ). The relationship between the gut microbiota and asthenozoospermia was examined using a Spearman correlation analysis to study the correlation between significantly different p_Bacteroidota, p_Firmicutes, g_Dubosiella, and g_Faecalibaculum with the major parameters of asthenozoospermia ( Figure 2G ). The results showed that p_Bacteroidota and g_Faecalibaculum were negatively correlated with the sperm concentration and motility (P < 0.01, P < 0.05), while p_Firmicutes and g_Dubosiella were positively correlated with the sperm concentration and motility (P < 0.01, P < 0.05).

Gut microbiota balance and health are crucial to maintain intestinal barrier integrity. An adverse gut microbiota structure can disrupt the intestinal barrier and cause various diseases (23). The H&E staining revealed that the intestinal barrier in the colonic tissue of the model group mice was disrupted, and this was accompanied by varying degrees of inflammatory infiltration ( Figure 3A ). We further evaluated the protein and mRNA expression levels of Occludin, ZO-1, and Claudin-1 using western blotting and real-time PCR, respectively ( Figures 3B, C ). The results showed that the mRNA and protein expression levels of Occludin, ZO-1, and Claudin-1 were significantly downregulated in the model group (P < 0.01, P < 0.05). However, these changes were reversed after acupuncture treatment (P < 0.01, P < 0.05), indicating that acupuncture can improve impaired intestinal barrier function.

Gut barrier disruption can cause the movement of harmful gut microbes across the barrier into the bloodstream. This triggers inflammatory responses and immune system dysregulation, altering the composition of plasma metabolites (24). Therefore, we analyzed the metabolites in the serum of each group. We performed classification statistics on all identified metabolites based on their chemical classification information. This analysis allowed us to display the proportion of each metabolite category using pie charts. The results indicated that under both ESI+ and ESI– modes, the metabolites were primarily composed of lipids and lipid-like molecules and organic acids and derivatives ( Figure 4A ). OPLS-DA model were then used to detect intergroup differences. Figures 4B, C shows the comparisons between the control group and the model group, as well as between the model group and the ACU group. There were significant separations between these groups, while the distribution points within each group were clustered. In addition, the internal cross-validation results of this model indicated that in the ESI+ mode, between the control group and the model group, R2Y = 0.997 and Q2Y = 0.830, and between the model group and the ACU group, R2Y = 0.994 and Q2Y = 0.927. In the ESI- mode, between the control group and the model group, R2Y = 0.996 and Q2Y = 0.846, and between the model group and the ACU group, R2Y = 0.999 and Q2Y = 0.863. These results suggested that the model had strong interpretability and good predictability. We also conducted permutation tests as an external validation method to further validate the model reliability. Figures 4B, C also shows that all of the Q2 points were lower than the rightmost original Q2 point. This indicated that the results of this study were reliable and effective.

In the combined ESI+ and ESI– modes, 112 DEMs were identified between the control group and the model group (52 upregulated and 60 downregulated), and 58 DEMs were identified between the model group and the ACU group (20 upregulated and 38 downregulated) ( Supplementary Table 1 ). The clustering heatmap showed significant differences between the control group vs. model group and model group vs. ACU group ( Figure 4D ), meeting the criteria for further analysis. A Venn diagram was drawn ( Figure 4E ), and we found that five DEMs were upregulated in the model group and downregulated after acupuncture treatment, while five DEMs were downregulated in the model group and upregulated after acupuncture treatment ( Figure 4F ). These 10 DEMs may be important targets through which acupuncture improves the condition, and they were then used in the subsequent functional enrichment analysis. The KEGG analysis revealed that these DEMs were primarily associated with pathways such as pyrimidine metabolism, protein digestion and absorption, and ABC transporters. ( Figure 4G ). A Spearman correlation analysis was performed to explore the relationships between the 10 DEMs and the key parameters of asthenozoospermia, as well as the gut microbiota ( Figure 4H ). Cytosine, 2’-deoxycytidine, 5-methylcytosine, 5-methyl-2’-deoxycytidine, and 4-aminophenol all showed a positive correlation with the sperm parameters or serum hormone levels (P < 0.01, P < 0.05). N-oleyl-leucine was negatively correlated with these sperm parameters or serum hormone levels (P < 0.01). In addition, several metabolites exhibited significant correlations with the gut microbiota (P < 0.01, P < 0.05).

Disruption of the BTB integrity is a significant cause of impaired spermatogenesis (25). We evaluated the expressions of the key BTB proteins, Occludin, Connexin-43, and N-cadherin, using western blotting to investigate the acupuncture effect on BTB in the asthenozoospermic mice ( Figure 5A ). We observed the distribution of these proteins within the testes using immunofluorescence ( Figures 5B–E ). The results showed that the positive signal for the tight junction protein Occludin was primarily expressed at the tight junctions formed by the Sertoli cells, particularly in the BTB region of the seminiferous tubules. Connexin-43 positive signals were detected in the spermatogenic cells, Sertoli cells, and the surrounding interstitial cells of the testes. N-cadherin was primarily expressed in the contact areas between the Sertoli cells and spermatogenic cells. The protein expressions of Occludin, Connexin-43, and N-cadherin were reduced to varying degrees after the cyclophosphamide treatment (P < 0.01). This result indicated that the BTB integrity was compromised. However, acupuncture treatment mitigated the reduction trend in the protein levels of Occludin and Connexin-43 (P < 0.01, P < 0.05), suggesting that acupuncture can protect BTB integrity.

Harmful substances and immune cells may infiltrate the testes when the BTB is disrupted. This triggers local inflammation and immune responses that alter the metabolic environment within the testes. In addition, this disruption can impair the synthesis and secretion of testicular hormones. This will further interfere with normal metabolism (26). Therefore, we analyzed the metabolites in the testes of each group. All identified metabolites were categorized according to their chemical classifications. The results indicated that in both the ESI+ and ESI– modes, the metabolites predominantly belonged to four major categories: organic acids and derivatives, lipids and lipid-like molecules, organoheterocyclic compounds, and benzenoids, and they collectively accounted for approximately 80% of the total metabolites ( Figure 6A ). We then used the OPLS-DA model to detect the intergroup differences. The results showed an obvious separation between the control and model groups as well as between the model and ACU groups, and there existed a tight clustering of samples within each group ( Figures 6B, C ). The internal cross-validation of the model revealed that in the ESI+ mode, the R2Y and Q2Y values between the control and model groups were 0.998 and 0.863, respectively, and between the model and ACU groups they were 0.994 and 0.571, respectively. In the ESI– mode, the R2Y and Q2Y values between the control and model groups were 0.998 and 0.861, respectively, and between the model and ACU groups, they were 0.996 and 0.583, respectively. These findings suggested that the model had strong explanatory power but moderate predictability. We then conducted a permutation test to further validate the model reliability. All of the Q2 points were lower than the original Q2 point on the far right ( Figures 6B, C ), and most Q2 points intersected the vertical axis at values less than zero. These findings indicated that the results of this study were reliable and valid.

In the combined ESI+ and ESI– modes, 457 DEMs were identified between the control and model groups (269 upregulated and 188 downregulated), and 178 DEMs were identified between the model and ACU groups (137 upregulated and 41 downregulated) ( Supplementary Table 2 ). The clustering heatmap revealed distinct differences between the control vs. model groups and the model vs. ACU groups ( Figure 6D ). These findings indicated that the results were reliable and suitable for further analysis. The Venn diagram ( Figure 6E ) showed that 22 DEMs were upregulated in the model group and downregulated after acupuncture treatment, while 71 DEMs were downregulated in the model group and upregulated after acupuncture treatment. In addition, we highlighted the top 10 DEMs with the highest VIP values ( Figure 6F ). The KEGG functional enrichment analysis of these 93 intersecting DEMs revealed that they were primarily associated with metabolic pathways such as ABC transporters, neuroactive ligand–receptor interaction, and the biosynthesis of amino acids ( Figure 6G ). A Spearman correlation analysis was conducted to investigate the relationships between the top 10 testicular DEMs that had high VIP scores and the key parameters of asthenozoospermia, the gut microbiota, and the serum DEMs ( Figure 6H ). 5-fluorouridine and 1-acetylimidazole were strongly negatively correlated with the sperm parameters and the serum hormone levels (P < 0.01). By contrast, the remaining testicular DEMs exhibited strong positive correlations with these parameters (P < 0.01). In addition, most testicular DEMs showed varying degrees of correlation with the serum DEMs (P < 0.01, P < 0.05).

We conducted further verification using an FMT experiment to determine whether the reproductive protective effects of acupuncture on the asthenozoospermic mice were dependent on the gut microbiota ( Figure 7A ).Compared with the FMT-M group, the FMT-A group showed varying degrees of improvement in testicular weight, testicular index, sperm concentration, sperm motility, and serum levels of T and LH (P < 0.01, P < 0.05). These results were consistent with the results observed in acupuncture-treated mice ( Figures 7B–D ). In addition, the FMT-M group had a lower sperm count and abnormal morphology that included irregular head size and shape, abnormal tail bending or folding, and these could all potentially affect sperm motility and fertilization capability (27). The testes of the FMT-M group also showed significant morphological abnormalities and structural disorganization. By contrast, the sperm and testicular morphology and structures of the FMT-A group improved to varying degrees ( Figures 7E, F ). In conclusion, these results suggested that the reproductive protective effects of acupuncture can be achieved using FMT.