Effects of PM2.5 Metal Components Derived From Porcine Farm Exposure on Sperm Function in Mice

ABSTRACT

This study aimed to identify the effects of major metal components present in particulate matter (PM)2.5 on the reproductive system, sperm function, and embryo development. Through intratracheal instillation, male mice were exposed to various concentrations of metal components, including calcium oxide (Ca), iron oxide (Fe), aluminum oxide (Al), zinc oxide (Zn), lead oxide (Pb), and a mixture of these metals, in PM2.5 collected from the porcine farm. After 14 days, testicular inflammation and abnormal sperm morphology were observed in the exposed mice. These results indicate that such metal exposure enhances inflammatory cytokines in the testis and oxidative stress‐induced apoptosis. Moreover, the exposure influenced sperm deformation, capacitation status, testosterone levels, and testosterone biosynthesis. Importantly, embryo development was also found to be impacted due to decreased sperm fertility. This study demonstrates that major metal components of PM2.5 derived from porcine farm pose adverse effects on the male reproductive system.

Exposure to PM2.5 metal components (Ca, Fe, Al, Zn, Pb) from pig farm negatively impacts male reproductive health in mice, causing reduced sperm quality, decreased testosterone levels, and impaired embryo development. This highlights potential reproductive risks for both animals and humans in agricultural environments.

1. Introduction

Particulate matter (PM) has emerged as a global environmental concern, exacerbated by rapid industrial development, posing significant public health burdens by contributing to various diseases [1, 2]. The multifaceted composition of PM, shaped by geographic, seasonal, and temporal factors, highlights the complexity of this environmental challenge [3, 4, 5, 6, 7]. Despite extensive research on PM sources and their impact on health, the effects of PM from agriculture and livestock farms remain relatively unexplored [8, 9].

Studies have revealed that elevated levels of PM2.5 in pig farms consist of inhalable and respirable particles that induce immune responses and adverse health effects in pigs [10, 11]. The contaminated conditions in farms, often comprising excrement, litter, feed, skin, and feathers, contribute to the formation of PM with varying sizes, compositions, and primary constituents compared to general PM [12, 13, 14]. In particular, PM in pig farms has been shown to comprise metal oxides, organic compounds, inorganic carbonaceous material, and ions such as ammonia (NH3) [15, 16, 17]. It has been demonstrated that exposure to specific metal‐based particulates, such as nickel and titanium nanoparticles (NPs), can potentially cause DNA damage and reproductive toxicity [13, 18, 19]. A previous study has demonstrated that five major metals (Ca, Fe, Zn, Pb, and Al) in PM2.5 from porcine farm induce lung inflammation, systemic inflammation, and body fluid imbalance in mice [20].

Recent research emphasizes the need for a more detailed understanding of the health impacts of PM exposure associated with agricultural activities [21, 22]. For this, it is important to consider the dynamic nature of PM composition, which is influenced by microbial interactions, climate change, and land‐use patterns [8, 23, 24]. Additionally, there is a growing interest in the interplay between PM exposure and genetic susceptibility, as individual variabilities may influence its impact on health [25]. Exposure to PM during oocyte maturation induces cell cycle arrest, reactive oxygen species (ROS) generation, and early apoptosis in mice [26]. Fine PM2.5 exposure adversely affects ovarian function and embryo quality [27], while PM2.5 impairs sperm quality and testosterone production in mice [28]. However, the specific nature and toxicity of major metal components in PM from porcine farms, particularly their impact on the reproductive system and underlying mechanisms, remain poorly understood.

Heavy metals in PM2.5 from pig farms primarily derive from feed and manure [14]. These metals are highly toxic due to the microbial activity arising from the fermentation of manure and residuals [29]. Furthermore, they contribute to the formation of PM with varying sizes, such as PM10 and PM2.5 [30]. Particularly toxic heavy metals include arsenic (As) [31, 32], cadmium (Cd) [33, 34, 35], and mercury (Hg) [36], which pose significant threats to biological systems by inducing infertility, depending on the environmental and occupational settings [37].

In this study, we identified the five primary metal components in PM2.5 collected from a porcine farm. Each metal was individually evaluated for its impact on the male reproductive system in mice [38], as these metals can persistently accumulate in the reproductive organs of pigs and farmers, potentially causing various health issues. The identified metal components, including calcium oxide (Ca), iron oxide (Fe), aluminum oxide (Al), zinc oxide (Zn), lead oxide (Pb), and a mixture of these metals, were administered to mice via intratracheal instillation. After 14 days, we investigated the impact of such exposure on the reproductive system, sperm function, and embryo development. This study highlights the potential hazards of metal components in PM2.5 from porcine farms and their multifaceted impact on male reproductive health, providing essential insights to guide policies and interventions to mitigate the adverse effects of agricultural PM exposure.

2. Materials and Methods

2.1. Metal Components in PM2.5

A sample of PM was collected from a porcine farm located in Iksan‐si, Jeollabuk‐do, Republic of Korea. The sample was analyzed using the PM2.5 Air Sampler (25‐mm PVC filter; AIR METRICS, Eugene, OR, USA) on October 28, 2021. Inductively coupled plasma‐mass spectrometry (ICP‐MS) was used to identify the metal composition in the sample: 100 μg/m³ of metal oxides in PM2.5 contained 48.3 μg/m³ Ca, 3.9 μg/m³ Fe, 1 μg/m³ Al, 0.9 μg/m³ Zn, and 0.3 μg/m³ Pb, yielding a total of 54.5 μg/m³ of mixed metals (Table 1). All identified metals, including Ca (634182‐25 G), Fe (Ⅱ, Ⅲ, 637106‐25 G), Al (544833‐10 G), Zn (544906‐10 G), and Pb (241547‐100 G) were purchased from Sigma‐Aldrich (St. Louis, MO, USA). These metals were dispersed in ultrapure water and subjected to 20 min of sonication to ensure proper suspension before the experiment [20].

Table 1.

Concentration of major metal components in PM2.5 collected from the porcine farm.

Components	μg/m3
Ca	48.3
Fe	3.9
Al	1.0
Zn	0.9
Pb	0.3
Mix	54.5

Note: Concentrations of major metal components (μg/m³) detected in PM2.5 samples collected from the porcine farm. The “Mix” represents the total concentration of all measured metal components.

2.2. Animal Experiment

All animal experiments were approved by the Institutional Animal Care and Use Committee of Kyungpook National University, according to the animal welfare and ethical guidelines (approval no. 2022‐0387). All mouse care and experimental procedures followed the ARRIVE guidelines (https://arriveguidelines.org). A total of 42 ICR mice (11 weeks old, male) were obtained from SAMTACO BIO KOREA Company (Osan‐si, Republic of Korea). The mice were housed at 18°C–25°C under a 12‐h light/dark cycle with unrestricted access to food and water. The mice were randomly divided into seven groups (n = 6/group). The mice were acclimatized for 1 week before the experiment. The control group received ultrapure water, while the other groups were exposed to specific concentrations of Ca, Fe, Al, Zn, Pb, or their mixture.

Intratracheal instillation was performed using a 24 G catheter, with the volume of metal suspension or ultrapure water kept below 50 μL, followed by 1 min of air supply [39, 40, 41, 42]. The mice were monitored to ensure their airways were not obstructed after instillation [20]. The mice were placed in cages for further monitoring. Body weights of the exposed mice were measured weekly, and on the final day, both body and testis weights were measured. The weight index was calculated using the following equation:

$$[\text{testisweight}(\mathrm{g})/\text{bodyweight}(g)]\times 100$$

After 14 days, the mice were anesthetized via intraperitoneal injection of 2,2,2‐tribromoethanol (T48402; Sigma‐Aldrich) dissolved in 2‐methyl‐2‐butanol (152463; Sigma‐Aldrich). Blood, testes, and cauda epididymis were collected for analysis.

2.3. Histopathological Analysis

The left testes were fixed in 4% paraformaldehyde in phosphate‐buffered saline (PBS) and then embedded in paraffin blocks. The samples were sectioned at a 4‐µm thickness and stained with hematoxylin and eosin (H&E). These sections were observed under a light microscope and analyzed using LAS AF Ink (Leica Microsystems GmbH, Wetzlar, Germany). ImageJ (National Institutes of Health, Bethesda, MD, USA) was used to measure the diameters of the seminiferous tubules and their lumens.

2.4. Real‐Time Polymerase Chain Reaction (RT‐PCR)

The messenger RNA (mRNA) expression levels of inflammation, oxidative stress, and testosterone biosynthesis markers in the collected testes were measured by RT‐PCR. RNA was extracted from the testes using TRI‐solution (TS200‐001; Bio Science Technology, Daejeon, Republic of Korea). Complementary DNA (cDNA) synthesis was performed using PrimeScript™ 1st Strand cDNA Synthesis Kit (6110; Takara Biotechnology Co. Ltd., Shiga, Japan). RT‐PCR was conducted using 8 μL of cDNA, 10 μL of Power SYBR®‐Green PCR Master Mix (4367659; Thermo Fisher Scientific, Waltham, MA, USA), 1 μL of forward primer (0.2 pmol), and 1 μL of reverse primer (0.2 pmol) (Table 2).

Table 2.

Primer sequences used for PCR analyses.

Category	Primer	Forward	Reverse
	β‐actin	GCG CAA GTA CTC TGT GTG GA	ACA TCT GCT GGA AGG TGG AC
Inflammation	Interleukin (IL) ‐6	GTT GTG CAA TGG CAA TTC TGA	TTG GTA GCA TCC ATC ATT TCT TTG
	Interleukin (IL) ‐1β	CCC CAG GGC ATG TTA AGG A	TGA CCC TGA GCG ACC TGT CT
	Tumor necrosis factor‐α (TNF‐α)	GCT GAG CTC AAA CCC TGG TA	CGG ACT CCG CAA AGT CTA AG
Antioxidant	Catalase	CGA CCA GGG CAT CAA AAA CT	ATT GGC GAT GGC ATT GAA A
	Superoxide dismutase (SOD)‐1	GAC TTG GGC AAA GGT GGA AA	CAG GGA ATG TTT ACT GCG CAA T
Testosterone biosynthesis	Steroidogenic Acute Regulatory Protein (StAR)	GAA CGG GGA CGA AGT GCT AA	TGG TCT ACC ACC ACC TCC AA
	Cholesterol side‐chain cleavage enzyme (P450scc)	GGA CAG TAT GCT GGC TAA AGG A	CGT AGG GCT CAG GAA AGG TTG
	3β‐Hydroxysteroid dehydrogenase (3β‐HSD)	TGG CAA CGA GGA AGA GCA TC	TTG CCC GTA CAA CCG AGA ATA
	17β‐Hydroxysteroid dehydrogenase (17β‐HSD)	ACA AGA TGA CCA AGA CCG CC	CCA CAG GAT TCA GCT CCG AT

Note: Primer sequences used for PCR analyses targeting genes related to inflammation, antioxidant defense, and testosterone biosynthesis in mice. β‐actin was used as the housekeeping gene. Forward and reverse primer sequences are listed for each gene.

2.5. Western Blot

Proteins were extracted from the right testes using PRO‐PREP of Cell/Tissue Protein Extraction Solution Kit (17081; Intron Biotechnology, Kirkland, WA, USA) and Protease Inhibitor Cocktail (P3100‐001; GenDEPOT, Katy, TX, USA). Protein concentrations were calculated using BCA Protein Assay Reagent A (WF322788; Thermo Fisher Scientific) and Reagent B (23224; PIERCE, Kyiv, Ukraine). A total of 30 µg of protein was loaded and separated using 8%–12% sodium dodecyl‐sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) and then transferred to a polyvinylidene difluoride membrane [43]. The membrane was blocked with 5% skim milk in Tris‐buffered saline with 0.1% Tween 20 (TBST) and incubated with primary antibodies against β‐actin (sc‐47778; Santa Cruz Biotechnology, Dallas, TX, USA), catalase (sc‐271803; Santa Cruz Biotechnology), SOD1 (sc‐101523; Santa Cruz Biotechnology), Bax (sc‐7480; Santa Cruz Biotechnology), and Bcl‐2 (sc‐7382; Santa Cruz Biotechnology) dissolved in TBST with 3% skim milk. An ECL detection kit (12316992; GE Healthcare, Chicago, USA) was used to detect the protein expression levels using the Da Vinci Fluorescence Imaging System (Da Vinci‐K, Seoul, Republic of Korea). β‐Actin was used as the loading control.

2.6. Sperm Analysis

Sperm was collected from the epididymis of three mice per group. The collected sperm was incubated in 2 mL of M2 medium (M7167; Sigma‐Aldrich) supplemented with 0.05% penicillin‐streptomycin (Gibco™, Grand Island, NY, USA) in a 35‐mm petri dish at 37°C. The cauda epididymis was split to release sperm into the M2 medium. The sperm was incubated for 90 min to assess motility. The sperm count was determined using a hemocytometer. The sperm was stained with Eosin Y and covered with a coverslip. Abnormalities were checked across 400 sperm [44]. Sperm concentration was incubated for 90 min for activation.

A computer‐assisted sperm analysis (CASA) program (SAC5, HT CASA, Barcelona, Spain) was used to assess sperm motility, with the Makler counting chamber pre‐heated to 37°C [45]. Sperm motility was observed using a Nikon Eclipse E200 LED MV R microscope (Tokyo, Japan). Total motility (%) represents the percentage of moving sperm in the sample, while progressive motility (%) indicates the percentage of sperm swimming mostly in straight lines or large circles. Sperm motility was categorized into rapid motility (%), middle motility (%), and slow motility (%). Additional sperm motility parameters were measured: VAP (average path velocity), VSL (straight‐line velocity), VCL (curvilinear velocity), ALH (amplitude of lateral head displacement), BCF (beat cross frequency), LIN (linearity), STR (straightness), and WOB (wobble).

2.7. Assessment of Sperm Capacitation Status

Capacitation status was evaluated using a dual staining method, involving Hoechst 33258 (H33258) and chlortetracycline (CTC) [46]. Sperm was incubated for 90 min for activation and centrifuged for 3 min. The supernatant was removed, and the resulting pellet was then resuspended in PBS containing H33258 (10 μg/mL; H1343; Sigma‐Aldrich) and incubated for 3 min at room temperature. Next, 250 μL of 2% (w/v) polyvinylpyrrolidone in Dulbecco's PBS (DPBS) was added to the sperm suspension, followed by centrifugation for 5 min. The supernatant was discarded, and the sperm pellet was resuspended in 150 μL of CTC solution (750 mM CTC in 5 μL of buffer containing 20 mM Tris, 130 mM NaCl, and 5 mM cysteine, pH 7.4) and mixed with 150 μL of DPBS (1:1, v/v). The samples were incubated in the refrigerator while being protected from light. The sperm samples were placed on a slide and covered with a coverslip. Acrosome‐reacted sperm (AR), live capacitated sperm (B), live non‐capacitated sperm (F), and dead sperm (D) patterns were assessed across 400 sperm (Figure 4A). H33258 and CTC fluorescence signals were detected using a Leica DM 2500 fluorescent microscope (Wetzlar, Germany) equipped with an EL 6000 external light source. The sperm were then categorized into the described patterns.

Figure 4.

The effects of PM2.5 metal exposure on capacitation status. (A) Images showing AR (acrosome‐reacted), B (capacitated), F (non‐capacitated), and D (dead) patterns. (B) Capacitation status of the AR pattern (%). (C) Capacitation status of the B pattern (%). (D) Capacitation status of the F pattern (%). (E) Capacitation status of the D pattern (%). N = 3 per group. The metal‐treatment groups (Ca, Fe, Al, Pb, and metal mixture) compared with the control group: p < 0.05 *, p < 0.01 ** and p < 0.001 ***.

2.8. Serum Hormone Levels

Blood was collected from the abdominal aorta, centrifuged at 4000 x g for 30 min at 4°C, and stored at ‐80°C until experiments. The serum testosterone levels were assessed using a mouse/rat testosterone ELISA kit (ab285350; Abcam, Cambridge, UK) [18]. To each well containing the samples, 100 μL of enzyme conjugate was added. The absorbance at 450 nm was measured using a microplate reader.

2.9. Embryo Collection and Culture

Ten female ICR mice (8 weeks old) were obtained from SAMTACO BIO KOREA Company. The mice were housed at 18°C–25°C under a 12‐h light‐dark cycle with unrestricted access to food and water. Superovulation was induced in these mice via intraperitoneal administration of 5 IU of pregnant mare serum gonadotropin (PMSG; hor‐272‐b; Prospec International, Ness‐Ziona, Israel), followed by 5 IU of human chorionic gonadotropin (hCG; C1063‐1VL; Sigma‐Aldrich) 48 h later. These female mice were then mated with male mice exposed to the aforementioned metals. After 14 h, vaginal plugs were examined to confirm successful mating. The successfully mated females were sacrificed to collect embryos.

Zygotes were collected from the oviducts and treated with hyaluronidase (H4272‐30MG; Sigma‐Aldrich) in M2 medium (M7167; Sigma‐Aldrich) to remove cumulus cells. The zygotes were washed three times with M2 medium and then cultured in M16 medium (M7292; Sigma‐Aldrich) under oil (NidOil; NO‐400K, Nidacon International, Gothenburg, Sweden) at 37°C with 5% CO₂. After 4 days, the blastocyst‐stage embryos were evaluated using a light microscope (Leica Microsystems GmbH) (Figure 6A).

Figure 6.

The effects of PM2.5 metal exposure on embryo development in vitro. (A) Images showing embryo development: (a) control group and (b) metal‐mixture group. Scale bar = 75 μm. (B) Embryo development stages (%): red asterisk, two‐cell stage; yellow asterisk, morula stage; black asterisk, blastocyst stage. N = 5 per group. The metal‐treatment groups (Ca, Fe, Al, Pb, and metal mixture) compared with the control group: p < 0.05 *, p < 0.01 ** and p < 0.001 ***.

2.10. Statistical Analysis

All experiments were conducted in triplicate, and the resulting data were evaluated using one‐way ANOVA followed by the post‐hoc Tukey test using SPSS version 16.0 (SPSS Inc., Chicago, IL, USA). The Shapiro–Wilk test was conducted for normality, and variance analysis was performed using the F‐test for comparing two groups and Bartlett's test (corrected) for comparing three or more groups. A non‐parametric Kruskal‐Wallis test was used if the data were not normally distributed. The results are presented as mean ± standard error of the mean (SEM), with no asterisk indicating p > 0.05, * indicating p < 0.05, ** indicating p < 0.01, and *** indicating p < 0.001 for statistical significance levels.

3. Results

3.1. The Effects of PM2.5 Metal Exposure on the Reproductive Organ

The PM2.5 metals were administered to male mice by intratracheal instillation and their impact on body and testis weights was monitored over 2 weeks (Figure 1A). Body weights were measured on days 0, 7, and 14, while testis weights were measured only on day 14. No statistical differences in body or testis weights were observed across all metal‐treatment groups (Figure 1B,C). Furthermore, the metal‐treatment groups showed no statistically significant differences in the testes‐to‐body weight ratio compared to the control group (Figure 1D). These results are consistent with previous studies [47, 48] and suggest that these metals do not display apparent toxicities that significantly decrease body or testis weights.

Figure 1.

The effects of PM2.5 metal exposure on body and testis weights in mice. (A) Protocol for intratracheal instillation of PM2.5 metals. (B) Body weight (g) changes. (C) Testis (g) weight changes. (D) Testis‐to‐body weight ratio (%). N = 6 per group. The metal‐treatment groups (Ca, Fe, Al, Pb, and metal mixture) compared with the control group: p < 0.05 *, p < 0.01 ** and p < 0.001 ***.

3.2. The Effects of PM2.5 Metal Exposure on Inflammation, Cell Death, and Testis Morphology

The sectioned testes were stained with H&E to assess the effects on testis morphology. The control group displayed normal morphology, including healthy spermatogenic cells, Leydig cells, and seminiferous tubules (Figure 2A). In contrast, the metal‐treatment groups showed a reduction in spermatogenic cells and an increase in lumen size. The Ca group exhibited abnormal spermatogenic cells, along with an increase in intercellular space. Additionally, the group treated with the metal mixture showed abnormalities in the seminiferous tubules, including the presence of intercellular vacuoles (Figure 2A). The results indicated a significant decrease in the diameter of the seminiferous tubules and lumen size in the metal‐treatment groups (p < 0.001) (Figure 2B,C), suggesting that metal exposure induced histopathological changes in the testis.

Figure 2.

The effects of PM2.5 metal exposure on histopathological lesions, inflammation, and apoptosis in the testis. (A) H&E‐stained sections of testes of metal‐exposed mice; black arrow, no lumen; blue arrow, Sertoli cell, and germ cell loss; red arrow, abnormal cell mass; scale bar = 80 μm. (B) Seminiferous tubule diameter measurements (μm). (C) Lumen size measurements (μm). (D‐F) Relative mRNA levels of inflammatory cytokines (TNF‐ α, IL‐1β, and IL‐6). (G‐H) Relative mRNA levels of antioxidant enzymes (catalase and SOD1). (I) Protein levels of antioxidant enzymes (catalase and SOD1) and (J) apoptosis markers (Bax and Bcl‐2). β‐actin was used as the loading control. N = 6 per group. The metal‐treatment groups (Ca, Fe, Al, Pb, and metal mixture) compared with the control group: p < 0.05 *, p < 0.01 ** and p < 0.001 ***.

We then measured the levels of inflammatory markers, including TNF‐α, IL‐1β, and IL‐6, in the testis. TNF‐α levels were significantly increased in the Fe, Al, Zn, and metal‐mixture groups compared to the control group (p < 0.01) (Figure 2D). IL‐1β levels were also significantly increased in the Fe, Al, and metal‐mixture groups (p < 0.05) (Figure 2E). IL‐6 levels were significantly elevated in the Fe and Al groups compared to the control group (p < 0.05) (Figure 2F). These findings suggest that metal exposure elevated inflammatory cytokine levels in the testis.

Given the increase in inflammatory cytokines, we next measured antioxidant levels to confirm oxidative stress caused by elevated reactive oxygen species (ROS) levels. The mRNA expression of catalase was significantly increased in the Ca, Fe, and Al groups (p < 0.05) (Figure 2G). Additionally, the mRNA expression of SOD1 was significantly elevated in all metal‐treatment groups (p < 0.05) (Figure 2H). These results were confirmed at the protein level, showing elevated catalase and SOD1 levels in the metal‐treatment groups (Figure 2I).

Apoptotic and antiapoptotic markers were also assessed at the protein level. Bax levels were increased in the metal‐treatment groups, while Bcl‐2 levels were decreased (Figure 2J). These results indicate that metal exposure elevates inflammatory cytokine levels, oxidative stress, and apoptotic cell death in the affected testes.

3.3. The Effects of PM2.5 Metal Exposure on Sperm Quality

We next assessed the changes in sperm phenotype following PM2.5 metal exposure. Sperm motility was found to be significantly affected, as analyzed using CASA (p < 0.05) (Table 3). Total motility was significantly reduced in the metal‐treatment groups compared to the control group (p < 0.001) (Figure 3A). Metal exposure also decreased the percentage of sperm showing progressive or rapid motility compared to the control group (p < 0.05) (Figure 3B,C). Accordingly, sperm displaying slow motility was significantly increased in the Al, Zn, Pb, and metal‐mixture groups compared to the control group (p < 0.05) (Figure 3D).

Table 3.

Effects of metal components exposure on sperm motility.

	Con	Ca	Fe	Al	Zn	Pb	Mix
Mot (%)	92 ± 1.25	80.75 ± 1.1*	40.20 ± 2.9***	70.30 ± 0.8***	80.08 ± 1.84***	73.85 ± 2.24***	80.2 ± 2.19***
Progressive (%)	54.24 ± 4.14	45.4 ± 2.97	14.68 ± 1.7***	36.55 ± 0.84***	38.97 ± 2.24*	38.14 ± 3.23*	32.77 ± 3.1**
Rapid (%)	71.71 ± 4.8	59.85 ± 1.03*	17.41 ± 2.86***	45.85 ± 1.06***	55.96 ± 2.89*	48.9 ± 1.78**	47.5 ± 2.7**
Medium (%)	8.5 ± 2.99	7.89 ± 0.43	7.23 ± 1.39	6.69 ± 1.23	6.94 ± 0.7	4.27 ± 0.64	7.72 ± 0.73
Slow (%)	11.79 ± 1.1	12.92 ± 1.51	15.56 ± 1.88	17.76 ± 1.4*	17.19 ± 1.51*	20.68 ± 0.9***	24.98 ± 2.65**
VCL (μm/s)	122 ± 9.15	120.63 ± 1.98	53.8 ± 13.52**	103.01 ± 4.26	114.28 ± 3.69	106.14 ± 0.89	103.9 ± 5.07
VSL (μm/s)	62.02 ± 4.53	59.17 ± 2.02	31.04 ± 8.86*	53.03 ± 4.92	51.85 ± 2.87	54.89 ± 3.26	45.7 ± 3.3*
VAP (μm/s)	75.5 ± 5.15	72.31 ± 2.24	37.25 ± 9.92*	63.58 ± 5.15	65.69 ± 3.36	66.44 ± 2.98	59.06 ± 3.26*
LIN (%)	50.84 ± 0.39	49.02 ± 0.88	56.12 ± 5.37	51.3 ± 3.19	45.35 ± 1.79*	51.69 ± 2.83	43.89 ± 1.22**
STR (%)	82.09 ± 0.43	81.81 ± 0.43	81.83 ± 3.59	83.23 ± 1.12	78.9 ± 0.75*	82.51 ± 1.3	77.22 ± 1.31*
WOB (%)	61.94 ± 0.6	59.92 ± 0.87	68.27 ± 3.64	61.57 ± 3.19	57.45 ± 1.79*	62.57 ± 2.46	56.83 ± 0.8**
ALH (μm)	5.10 ± 0.29	5.30 ± 0.04	2.32 ± 0.57**	4.87 ± 0.14	5.62 ± 0.17	5.11 ± 0.17	5.64 ± 0.06
BCF (Hz)	10 ± 0.54	10.11 ± 0.28	5.87 ± 1.01*	11.1 ± 0.34	10.51 ± 0.11	10.35 ± 0.38	10.13 ± 0.4

Note: Total motility (%): percentage of motile sperm in the entire sample. Progressive motility (%): percentage of sperm swimming mostly in a straight line or large circles. Rapid (%); Medium (%); Slow (%); VAP: average path velocity; VSL: straight line velocity; VCL: curvilinear velocity; ALH: amplitude of lateral head displacement; BCF: beat cross frequency; LIN: linearity (VSL/VCL); STR: straightness (VSL/VAP); WOB: wobble (VAP/VCL); N = 3 per group; control group compared with metal groups (Ca, Fe, Al, Pb, and mixed); p < 0.05 *, p < 0.01 **, and p < 0.001 ***.

Figure 3.

The effects of PM2.5 metal exposure on sperm motility and abnormality. (A) Total motility (%). (B) Progressive motility (%). (C) Rapid motility (%). (D) Slow motility (%). (E) Sperm count (× 10⁶/mL). (F) Sperm morphology: (a) normal sperm and (b, c) abnormal sperm. (G) Abnormal sperm (%). N = 3 per group. The metal‐treatment groups (Ca, Fe, Al, Pb, and metal mixture) compared with the control group: p < 0.05 *, p < 0.01 ** and p < 0.001 ***.

VCL, VSL, VAP, ALH, and BCF values were decreased in the Fe group compared to the control group. The Zn group showed decreased LIN, STR, and WOB, while the metal‐mixture group exhibited decreased VSL, VAP, LIN, STR, and WOB values compared to the control group. These results suggest that metal exposure negatively impacts sperm motility in mice.

The Fe, Al, Pb, and metal‐mixture groups showed significantly reduced sperm count compared to the control group (p < 0.05) (Figure 3E), indicating the adverse effects of metal exposure.

Abnormal characteristics, such as bent heads, missing heads, bent necks, bent tails, and coiled tails, were observed in both the control and metal‐treatment groups. However, the metal‐treatment groups showed a significantly higher number of abnormal sperm compared to the control group (p < 0.001) (Figure 3F,G), indicating that metal exposure induces sperm deformation in mice.

3.4. The Effects of PM2.5 Metal Exposure on the Capacitation Status

To determine the impact on sperm fertility, the capacitation status was evaluated via the acrosome reaction. In the metal‐treatment groups, the number of sperm displaying the AR, B, and F patterns was significantly reduced compared to the control group (p < 0.05) (Figure 4B–D). Additionally, the number of sperm exhibiting the D pattern was significantly increased in the metal‐treatment groups compared to the control group (p < 0.001) (Figure 4E). These findings demonstrate that exposure to PM2.5 metals negatively affects the sperm capacitation status in mice.

3.5. The Effects of PM2.5 Metal Exposure on the Serum Testosterone Levels

Metal exposure was found to affect testosterone synthesis, likely due to increased inflammation and oxidative stress. Serum testosterone levels were significantly decreased in the Fe, Al, and metal‐mixture groups (p < 0.05) (Figure 5A), indicating that exposure to PM2.5 metals reduces testosterone levels. However, no statistically significant differences were observed in StAR levels between the metal‐treatment and control groups (Figure 5B).

Figure 5.

The effects of PM2.5 metal exposure on testosterone levels and testosterone biosynthesis‐related markers. (A) Testosterone level (ng/mL). Relative mRNA levels of (B) StAR, (C) P450scc, (D) 3β‐HSD, and (E) 17β‐HSD. N = 6 per group. The metal‐treatment groups (Ca, Fe, Al, Pb, and metal mixture) compared with the control group: p < 0.05 *, p < 0.01 ** and p < 0.001 ***.

The relative P450scc expression levels were significantly decreased in the Fe, Al, Pb, and metal‐mixture groups compared to the control group (p < 0.05) (Figure 5C). Additionally, 17β‐HSD levels were reduced in the Ca and Pb groups compared to the control group (p < 0.05) (Figure 5D). The 3β‐HSD expression levels were significantly decreased in the Al group (p < 0.05) (Figure 5E).

These results suggest that exposure to PM2.5 metals inhibits testosterone levels and testosterone biosynthesis in Leydig cells in mice.

3.6. The Effects of PM2.5 Metal Exposure on Embryo Development In Vitro

The obtained results showed that PM2.5 exposure significantly reduces sperm fertility, suggesting that embryo development may be affected as well. To investigate this phenomenon, the metal‐exposed male mice were mated with superovulated female mice. After successful mating, zygotes were collected from the oviducts and cultured in vitro to assess embryo development. The metal‐mixture group exhibited a significantly reduced number of blastocyst‐stage embryos compared to the control group, showing arrest at the two‐cell embryo stage (p < 0.05) (Figure 6B). These findings suggest that PM2.5 metal exposure hinders embryo development due to factors such as abnormal sperm phenotypes, low capacitation rates, and reduced testosterone levels.

4. Discussion

This study is the first to investigate how metals in PM2.5 prevalent in porcine farm affect the male reproductive system and embryo development in mice. The primary metal components in PM2.5 were identified to be metal oxide nanoparticles (MONPs) comprising Ca, Fe, Zn, and Pb. MONPs can infiltrate the epithelial barrier, placental barrier, and blood‐testis barrier (BTB), leading to their accumulation in reproductive organs [49]. Previous studies have demonstrated that these NPs enhance inflammatory responses and oxidative stress, resulting in ROS damage and cytotoxicity at both molecular and genetic levels [50]. Ironically, some of the metal components in MONPs are incorporated into various nutritional supplements and prominently consumed.

It has been suggested that the most abundant metals or high PM concentrations induce reproductive dysfunction through respiratory pathways [51, 52]. In particular, Pb exposure has been reported to induce toxic effects on the male reproductive system [53]. Specifically, heavy metals have been shown to enhance tissue oxidative stress, alter testicular histology, and elevate testicular apoptosis in rams [54]. Despite significant evidence of the toxic effects of metal components in PM2.5 prevalent in porcine farm, their specific effects on the reproductive system have remained unexplored thus far. In this regard, this study highlights the potential risks these metals pose to both farmers and animals. Our findings indicate that PM2.5 exposure may adversely affect male reproductive health.

A crucial finding of this study is the precise quantification of metal component concentrations in porcine‐farm PM2.5. The PM sample was collected in an enclosed area over a day, and the concentrations were calculated per unit area. The calculated metal concentrations were appropriately scaled and proportionally divided for administration to mice. Each metal or metal mixture was delivered via intratracheal instillation, a commonly used administration route alternative to inhalation that allows for precise dosage control and direct delivery of agents to the intratracheal region [55, 56].

Exposure to these metals did not significantly decrease body or testes weights in mice (Figure 1), which was consistent with previous studies [47, 48, 57]. However, metal exposure induced abnormalities in testis morphology, such as enlarged lumens, abnormal cell masses, and disorganized germinal epithelium (Figure 2A), likely due to oxidative stress and apoptosis (Figure 2G–J). As aforementioned, MONPs can cross BTB and accumulate in the testis, potentially disrupting BTB integrity and causing testicular damage [58]. Specifically, Ca exposure has been shown to inhibit spermatogenesis [59], cause cell apoptosis, and decrease semen quality, leading to male sterility [58, 60]. Heavy metals have also been linked to toxic effects on sperm motility in mice [61, 62]. For example, cobalt exposure significantly reduces sperm quality, count, and production, causing sperm abnormalities [63]. Additionally, Fe‐induced inflammation, oxidative stress, and apoptosis have been suggested to inhibit testosterone biosynthesis [64]. Unlike previous studies that frequently used high metal concentrations to assess toxicity, our study evaluated metal toxicity at levels commonly found in porcine farm, confirming the association of PM2.5 with testis abnormalities.

Excessive inhalation of Fe, known to induce oxidative damage, has been linked to inflammation and oxidative stress, leading to reduced sperm quality and quantity (Figure 2). This phenomenon can lead to abnormal sperm motility and disrupted spermatogenesis. High Fe concentrations have been associated with oxidative stress in seminal plasma and reduced sperm quality in humans [65]. Our findings directly demonstrated the relationship between Fe and testicular dysfunction caused by inflammation, oxidative stress, and abnormal sperm motility.

We observed that exposure to porcine‐farm PM2.5 metals did not significantly reduce sperm count in mice (Figure 3E). While zinc NPs have been shown to have minimal impact on sperm motility, concentration, and viability [66], our findings suggest that they may protect sperm production through their antioxidant properties. Previous studies on birds have shown improved sperm production following Ca supplementation [67]. In our study, while sperm production remained unchanged in the Ca‐exposed group, a reduction in sperm motility was observed.

Capacitation status is a key factor in sperm fertility. Under normal conditions, capacitated sperm change morphology and motility. Protein kinase A (PKA) activation and tyrosine phosphorylation in spermatozoa lead to capacitation and induce the acrosome reaction, allowing sperm to penetrate the zona pellucida during fertilization [68]. In our study, capacitated sperm exposed to metals showed significantly reduced AR, B, and F patterns, along with a significant increase in the D pattern (Figure 4), indicating that metal exposure impairs sperm fertility.

Testosterone is crucial for sperm production and the development of immature sperm. Our findings indicated that exposure to Fe, Al, or metal mixture significantly reduces serum testosterone concentrations and the enzymatic activities of p450scc, 3β‐HSD, and 17β‐HSD (Figure 5). This suggests that metal exposure disrupts testosterone biosynthesis, potentially causing endocrine‐disrupting effects. Testosterone biosynthesis is a steroid production process that begins with cholesterol in Leydig cells. The steroidogenic pathway, involving StAR, P450scc, 17β‐HSD, and 3β‐HSD, was analyzed to assess the level of cholesterol transport and testosterone biosynthesis in male mice [69]. StAR and P450scc are rate‐limiting enzymes in steroid hormone biosynthesis, with StAR transporting cholesterol to the mitochondrial membrane and P450scc converting cholesterol to pregnenolone [70]. We observed that exposure to Fe, Al, Pb, and metal mixture reduced P450scc expression levels (Figure 5C). 3β‐HSD and 17β‐HSD are enzymes in the smooth endoplasmic reticulum that convert steroid precursors into active hormones [71]. Previous studies have shown that exposure to nickel chloride and PM2.5 reduces StAR, P450scc, 17β‐HSD, and 3β‐HSD expressions [69]. We observed that Al exposure decreased 3β‐HSD levels (Figure 5D), while Ca and Pb exposure reduced 17β‐HSD levels (Figure 5E). These findings indicate that PM2.5 metals disrupt testosterone biosynthesis, particularly in Leydig cells, ultimately affecting spermatogenesis and sperm production.

In mice, spermatogenesis typically takes about 35 days [72], whereas in pigs, it lasts approximately 4 weeks [73]. Given that the 14‐day treatment period conducted in this study corresponds to approximately 40% of the spermatogenesis cycle in mice, an equivalent treatment duration in pigs would be around 4–5 weeks, as 14 days represent roughly 25% of the spermatogenesis cycle in pigs. The longer sperm maturation process and differing physiological conditions may contribute to variations in the impact of heavy metals on sperm. Although this study focused on male mice, future research should explore other animals, such as pigs, to more comprehensively understand the broader impact of PM2.5 exposure on reproductive health.

This study highlights the complex effects of metal components in PM2.5 prevalent in porcine farm on the male reproductive system and embryo development. The findings underscore the need for further research to unravel the molecular mechanisms underlying these effects, considering the dynamic nature of metal composition and their potential impact on human and animal fertility. Additionally, exploring interventions and protective measures against the adverse effects of metal exposure in agricultural contexts will be crucial.

5. Conclusion

In this study, the effects of exposure to the major metal components in PM2.5 prevalent in porcine farm on sperm function and the male reproductive system were investigated. Male mice were administered with the identified metals via intratracheal and assessed for their impact on reproductive health. The findings indicate that general PM inhalation during agricultural activities can adversely affect the male reproductive system. Further research is needed to elucidate the mechanisms underlying PM2.5‐induced alterations in sperm motility and reproductive toxicity in mammals.

Author Contributions

Chae Yeon Kim: conceptualization, writing – original draft. Chae Rim Kim: conceptualization, writing – original draft. Eungyung Kim: conceptualization, writing – original draft. Kanghyun Park: methodology. Hyeonjin Kim: methodology. Lei Ma: software. Ke Huang: methodology, validation. Zhibin Liu: validation. Junsu Park: investigation. Minwoong Jung: investigation. Shengqing Li: formal analysis. Weihong Wen: formal analysis. Sangsik Kim: data curation. Sijun Park: data curation. Zae Young Ryoo: visualization. Junkoo Yi: conceptualization, writing – review and editing, supervision. Myoung Ok Kim: writing – review and editing, supervision.

Conflicts of Interest

The authors declare no conflicts of interest.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.