Porcine reproductive and respiratory syndrome virus infects the reproductive system of male piglets and impairs development of the blood–testis barrier

ABSTRACT

Porcine reproductive and respiratory syndrome virus (PRRSV) causes a highly contagious disease that threatens the global swine industry. Recent studies have focused on the damage that PRRSV causes to the reproductive system of male pigs, although pathological research is lacking. Therefore, we examined the pathogenic mechanisms in male piglets infected with PRRSV. Gross and histopathological changes indicated that PRRSV affected the entire reproductive system, as confirmed via immunohistochemical analysis. PRRSV infected Sertoli cells and spermatogonia. To test the new hypothesis that PRRSV infection in piglets impairs blood – testis barrier (BTB) development, we investigated the pathology of PRRSV damage in the BTB. PRRSV infection significantly decreased the quantity and proliferative capacity of Sertoli cells constituting the BTB. Zonula occludens-1 and β-catenin were downregulated in cell – cell junctions. Transcriptome analysis revealed that several crucial genes and signalling pathways involved in the growth and development of Leydig cells, Sertoli cells, and tight junctions in the testes were downregulated. Apoptosis, necroptosis, inflammatory, and oxidative stress-related pathways were activated, whereas hormone secretion-related pathways were inhibited. Many Sertoli cells and spermatogonia underwent apoptosis during early differentiation. Infected piglets exhibited disrupted androgen secretion, leading to significantly reduced testosterone and anti-Müllerian hormone levels. A cytokine storm occurred, notably upregulating cytokines such as tumour necrosis factor-α and interleukin-6. Markers of oxidative-stress damage (i.e. H₂O₂, malondialdehyde, and glutathione) were upregulated, whereas antioxidant-enzyme activities (i.e. superoxide dismutase, total antioxidant capacity, and catalase) were downregulated. Our results demonstrated that PRRSV infected multiple organs in the male reproductive system, which impaired growth in the BTB.

Introduction

Porcine reproductive and respiratory syndrome (PRRS) is one of the most detrimental diseases in the global swine industry, being highly contagious and posing significant threats to profitability and the sustainability of the industry [1]. Despite extensive control measures, PRRS remains endemic in Asia, North America, and Europe [2]. Porcine reproductive and respiratory syndrome virus (PRRSV), the causative agent of PRRS, is an enveloped RNA virus classified in the Porartevirus genus, the Arteriviridae family, and the Nidovirales order [3]. Porcine alveolar macrophages are the primary target cells of PRRSV replication [4]. Currently, no fully effective vaccines or antiviral treatments are available against PRRSV, highlighting the importance of studying PRRSV – host interactions [5]. The pathogenic effects of PRRSV on the respiratory system and the reproductive performance of sows have been extensively studied [6–8]. In recent years, the level of interest in the impact of PRRSV on boar health and fertility has been growing.

In modern pig farming, the development of the boar reproductive system is crucial for sustainable production. In China, artificial-insemination techniques can result in over 3,000 piglets per boar annually, directly influencing fertility and the overall efficiency of pig production, resulting in significant economic value [9,10]. Thus, the growth and function of the reproductive system are crucial from birth to sexual maturity.

The boar reproductive system includes both external and internal genitalia. The external genitalia included the scrotum and penis. The internal male reproductive system includes the testes, epididymides, vas deferens, ejaculatory ducts, urethra, and accessory glands, such as the seminal vesicles, prostate gland, and bulbourethral glands [11,12]. Semen is a mixture of sperm and seminal plasma. Sperm cells are produced by the testes, mature in the epididymides, and are then transported through the vas deferens. Seminal plasma is primarily secreted by the accessory glands of the prostate gland, seminal vesicles, and bulbourethral glands [13]. The testes are key organs for male fertility and serve dual functions: they are the sites of both spermatogenesis and androgen production. The internal structure of the testis is divided into the interstitium and seminiferous tubules [14]. The interstitium is primarily composed of Leydig cells and macrophages, and it is responsible for testosterone (T) production and secretion [15]. Spermatogenesis occurs in the seminiferous tubules, which contain Sertoli cells that provide protection and nourishment for spermatogonia and spermatozoa [16]. Tight junctions (TJs) between Sertoli cells form the blood – testis barrier (BTB), which is one of the most crucial structures in the testis and is essential for maintaining an immunologically privileged environment [17,18]. The BTB separates the epithelial cells of the convoluted vasculature into a basal zone and a superior luminal zone, which isolates immunogenic developing germ cells from systemic circulation [19]. In addition, the BTB regulates the transport of substances into the lumen, creating a unique environment that is necessary for normal sperm development that also helps avoid triggering an autoimmune response and the production of anti-sperm antibodies [20]. If the BTB is compromised due to factors such as disease, injury, or inadequate nutrition during the developmental stage of the reproductive system, then it may have long-lasting effects on semen quality and fertility in adulthood for boars.

PRRSV infection poses a significant threat to the boar reproductive system [21]. Infection of the testes, epididymides, and bulbourethral glands has been reported, which potentially explains the presence of PRRSV in semen [22–24]. However, limited research has been conducted on the infection of other male reproductive organs, such as the seminal vesicles and prostate gland. Notably, PRRSV can infect Sertoli cells, which may compromise the TJ proteins responsible for maintaining the integrity of the BTB and cause harm to spermatogonia [25]. We hypothesized that such damage leads to persistent autoimmune responses, hormonal regulatory disorders, impaired spermatogenesis, and infertility in male pigs. Specifically, PRRSV infects piglets up to 30 days of age and such infections can cause irreversible damage to the male reproductive system. Further investigations are necessary to clarify the mechanisms underlying these infections and the associated damage.

A knowledge gap exists in terms of the effects of PRRSV on the BTB and its related mechanisms, particularly in young boars with immature reproductive functions. To fill this gap, we conducted experiments on 7-day-old male piglets and completely isolated the male reproductive system at 7 and 30 days after infection. Initially, we observed infection and damage to the organs and glands of the boar reproductive system following infection, and then we performed immunohistochemical (IHC) staining for further analysis. We focused on PRRSV infection of Sertoli cells (which form the BTB) and spermatogonia (which produce spermatozoa). Subsequently, we investigated the infection of Sertoli cells and disruption of the TJ caused by PRRSV via immunofluorescence (IF) staining for specific marker proteins. We comprehensively assessed testicular growth and development, apoptosis, inflammatory processes, hormone-secretion disorders, and oxidative-stress damage, and we clarified the relevant mechanisms of PRRSV-induced BTB disruption using transcriptomics. The outcomes of this study offer valuable insights for alleviating the negative effects of PRRSV on boar fertility and the overall herd health and for enhancing the productivity of the pig industry.

Materials and methods

Ethics statement

The animal experiments adhered to guidelines outlined by the Council of Agriculture Executive Yuan of the Republic of China. All animal experiments were approved by the Laboratory Animal Management Committee of Sichuan Province (approval number SYXK2019–187). All experimental procedures and animal-welfare standards strictly followed the Guidelines of Animal Management at Sichuan Agricultural University. Infection and euthanasia were performed under anaesthesia, and all possible measures were taken to minimize animal suffering.

Piglet-challenge test

We obtained 21-day-old male piglets from a farm in Chengdu. Before the study, the piglets tested negative for PRRSV, pseudorabies virus, porcine circovirus (types 2 and 3), classical swine fever virus, African swine fever virus, and porcine epidemic diarrhoea virus.

Male piglets were randomly assigned to four groups (five piglets/group) and housed under identical conditions with unrestricted access to food and water. Following a 24-hour acclimation period, two groups were intranasally administered 2 mL of the PRRSV SCABTC-202305 strain (genome sequence: GenBank accession number OR365672.1) stored in the Animal Biotechnology Center of Sichuan Agricultural University, with each mL containing 2 × 10⁵ 50% tissue culture infective doses. The two control groups received a sham inoculation with the same volume of Dulbecco’s modified Eagle’s medium (DMEM).

Rectal and testicular temperatures, testicular diameters, and survival rates were monitored daily after challenge with PRRSV or mock challenge with DMEM. Blood samples were collected on 0, 7, 14, 21, and 28 days post-challenge (dpc). Piglets in the PRRSV-inoculated and control groups were euthanized via intravenous injection with a lethal dose of sodium pentobarbital (Sinopharm) at 7 and 30 dpc. Immediately after euthanasia, necropsy was performed, and the complete male reproductive tract was extracted from each piglet. Excess tissues attached to the reproductive organs were removed and patted dry with blotting paper. Subsequently, the testes were isolated and weighed to the nearest mg. The tissue somatic indices (TSIs) were calculated using the following formula:

$$\mathit{TSI}=\frac{\mathit{Weight}\mathit{of}\mathit{the}\mathit{tissue}}{\mathit{Weight}\mathit{of}\mathit{the}\mathit{pig}}\times \mathit{\hspace{0.17em}}100\mathrm{\%}$$

Gross and microscopic lesion analysis

Reproductive organs (testes, epididymides, bulbourethral glands, prostate gland, and seminal vesicles) were fixed in 4% paraformaldehyde solution. The tissues were then dehydrated using an ethanol gradient, embedded in paraffin, and sectioned. The sections were deparaffinized, rehydrated, and stained with haematoxylin and eosin using standard protocols. The sections were visualized using a Pannoramic SCAN II slide scanner (3DHISTECH), and testicular development was histologically assessed. Statistical analysis of the diameters of the seminiferous cords/tubules was based on 250 randomly selected round cord/tubule cross-sections from each group, with 50 cross-sections per individual piglet.

Detecting viral loads

Total RNA was extracted from male reproductive-tract tissues and serum using the FastPure Viral DNA/RNA Mini Kit (Vazyme) following the manufacturer’s instructions. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis was performed to measure PRRSV-RNA levels. Briefly, 1 μg of total RNA was reverse-transcribed into complementary DNA (cDNA) using the PrimeScript^TM RT Reagent Kit (Takara). The sequences of the primers used were 5′-GCAAGTACATTCTGGCCCCT-3′ and 5′-CAATGTGCCGTTGACCGTAG-3′. The thermocycling conditions were as follows: 95°C for 30 s, followed by 40 cycles of 95°C for 5 s and 60°C for 20 s. PRRSV-RNA levels were normalized for relative quantification.

IHC analysis

IHC staining was performed on sections of the testes, epididymides, bulbourethral glands, prostate gland, and seminal vesicles. The tissue sections were treated with 3% H₂O₂ solution (pH 7.6) for 10 min and then with 5% bovine serum albumin (Thermo Fisher Scientific) for 30 min. The sections were then incubated overnight at 4°C with a primary rabbit monoclonal antibody against the PRRSV nucleocapsid protein (1:500; Chixun). After washing the sections thrice with phosphate-buffered saline (PBS), they were incubated at 37°C for 30 min with a secondary biotin-conjugated, affinity-purified goat anti-rabbit IgG antibody (Proteintech). After further incubation with streptavidin-biotin complexes (Boster) at 37°C for 30 min, specific binding was visualized using diaminobenzidine (Boster). The percentage of positive cells was calculated for each section using ImageJ software version 1.5.1 (National Institutes of Health).

IF analysis

Testicular sections were deparaffinized, rehydrated, and subjected to heat-mediated antigen retrieval in sodium citrate buffer (0.01 mol/L, pH 6.0). The testis sections were incubated with 10% donkey serum diluted in Dulbecco’s PBS for 2 h at 25°C. After blocking, the sections were incubated overnight at 4°C with primary antibodies diluted in 10% donkey serum. As primary antibodies, we used rabbit anti-SRY-box transcription factor 9 (SOX9; 1:100; ab185966, Abcam), mouse anti-proliferating cell nuclear antigen (PCNA; 1:800; BM0104, Boster), rabbit anti-zonula occludens-1 (ZO-1; 1:100; ab216880, Abcam), rabbit anti-β-catenin (1:200; 51067–2-AP, Protein Group), rabbit anti-CD86 (1:400; A00220–4, Boster), rabbit anti-CD163 (1:200; ab182422, Abcam), rabbit anti-interleukin-6 (IL-6; 1:100; BA4339, Boster), rabbit anti-tumour necrosis factor-alpha (TNF-α; 1:100; BS-2081 R, Bioss), and rabbit anti-interferon-gamma (IFN-γ; 1:100; DF6045, Addinity). After washing the testis sections, they were incubated for 1 h at 25°C with appropriate secondary antibodies diluted in 10% donkey serum. As secondary antibodies, we used an Alexa Fluor™ 594-conjugated donkey anti-rabbit IgG (H+L) highly cross-adsorbed secondary antibody (1:600; A21207, Thermo Fisher Scientific), an Alexa Fluor™ 488-conjugated donkey anti-rabbit IgG (H+L) highly cross-adsorbed secondary antibody (1:400; A21206, Thermo Fisher Scientific), an Alexa Fluor™ 594-conjugated donkey anti-mouse IgG (H+L) highly cross-adsorbed secondary antibody (1:400; A21203, Thermo Fisher Scientific), and a goat anti-rabbit IgG H&L (HRP) secondary antibody (1:4000; ab205718, Abcam). After washing, the sections were counterstained with 4′,6-diamidino-2-phenylindole (Abcam) for 5 min at 25°C, and microscopy was performed. To quantify the positive cells per seminiferous cord/tubule cross-section, we analysed 250 randomly selected round cord/tubule cross-sections from each group, with 50 cross-sections per individual piglet.

Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay

To study apoptosis, cells were stained using a TUNEL FITC Apoptosis Detection Kit (Vazyme).

Detecting cytokines in the testes and serum

Testicular samples were harvested, placed in 4°C in PBS, immediately transferred to a cold homogenizer, and centrifuged at 3000 rotations/min for 15 min. Each supernatant was transferred to an Eppendorf tube for further use.

Whole-blood samples were collected in tubes without anticoagulants and allowed to clot at 25°C for 30 min. The tubes were then centrifuged at 2000 × g for 10 min at 4°C. The serum supernatants were carefully aspirated and transferred to Eppendorf tubes for further use.

The levels of several cytokines (IFN-γ, IL-1α, IL-1β, IL-6, IL-10, and TNF-α) in piglet serum and testis samples were measured using Porcine ELISA Kits (Thermo Fisher Scientific).

Detecting hormone secretion in the testes

Serum T and anti-Müllerian hormone (AMH) levels were measured using Hormone Assay Kits (Nanjing JianCheng Bioengineering Institute).

Detection of oxidative stress and inflammatory responses in the testes

The H₂O₂, total antioxidant capacity (T-AOC), malondialdehyde (MDA), glutathione (GSH), oxidized glutathione (GSSG), superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-PX) levels in the testes were measured using specific assay kits for each analyse (Nanjing JianCheng Bioengineering Institute).

Transcriptome sequencing of testicles samples

RNA extraction

Total RNA was extracted from testis tissues as described above in the “Detecting viral loads” section. RNA quality was assessed using a 5300 Bioanalyzer (Agilent) and quantified using an ND-2000 instrument (NanoDrop Technologies). Only high-quality RNA samples with an optical density (OD)₂₆₀: OD₂₈₀ ratio of 1.8–2.2, an OD₂₆₀: OD₂₃₀ ratio of ≥ 2.0, an RNA integrity number of ≥ 6.5, a 28S: 18S RNA ratio of ≥ 1.0, and >1 μg sample obtained were used to prepare sequencing libraries.

Library preparation and next-generation sequencing

RNA purification, reverse transcription, library construction, and next-generation sequencing were conducted at Shanghai Majorbio Bio-pharm Biotechnology Co., Ltd. RNA-sequencing (RNA-seq) transcriptome libraries were prepared following the Illumina® Stranded mRNA Prep protocol, using 1 μg of total RNA and ligation reagents (Illumina). mRNA was isolated using the polyA-selection method with oligo(dT) beads and then fragmented using fragmentation buffer. Next, double-stranded cDNA was synthesized using a SuperScript Double-Stranded cDNA Synthesis Kit (Thermo Fisher Scientific) with random hexamers (Illumina). The synthesized cDNA was then subjected to end-repair, phosphorylation, and the addition of “A” bases following Illumina’s library-construction protocol. Libraries were chosen based on 300-base pair cDNA fragments, which were extracted from 2% low-range ultra-agarose gels. Subsequently, PCR amplification was performed for 15 cycles using Phusion DNA polymerase (Thermo Fisher Scientific). After quantification using a Qubit 4 Fluorometer (Thermo Fisher Scientific), each paired-end RNA-seq library was sequenced using a NovaSeq Xplus sequencer (Illumina) with a read length of 2 × 150 base pairs.

Quality-control analysis and read mapping

Raw paired-end reads were trimmed and quality-controlled using fastp software version 0.23.2 (HaploX) with the default parameters. Clean reads were aligned separately to the reference genome in orientation mode using HISAT2 software version 2.2.1 (Johns Hopkins University). The mapped reads from each sample were assembled using StringTie with a reference-based approach.

Differential expression and functional-enrichment analysis

To identify differentially expressed genes (DEGs) between two samples, transcript-expression levels were calculated using the transcripts per million reads method. RSEM software was used to quantify the gene abundances. We performed differential expression analysis using either DESeq2 or DEGseq. DEGs with a |log₂ fold-change| of ≥ 1 and a false-discovery rate FDR of < 0.05 (DESeq2) or < 0.001 (DEGseq) were considered significantly differentially expressed. We subjected the DEGs to functional-enrichment analysis using the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases to identify significantly enriched GO terms and metabolic pathways at a Bonferroni-corrected P-value of < 0.05, using the entire transcriptome background as a reference. GO functional-enrichment analysis and KEGG-pathway analysis were performed using Goatools and the Python SciPy library, respectively.

Alternative-splicing events

All alternative-splicing events that occurred in our samples were identified using the rMATS program. Only isoforms similar to the reference sequence or those containing novel splice junctions were considered. Splicing differences were detected in terms of exon inclusion; exon exclusion; and alternative 5′, 3′, and intron-retention events.

Validation of the RNA-seq results by RT-qPCR

To validate the accuracy of the RNA-seq data, we randomly selected eight mRNAs and confirmed their expression levels via RT-qPCR. Total RNA from testicular samples of infected and control piglets was extracted using the TRIzol Reagent (Invitrogen) and reverse transcribed using the PrimeScript™ RT Reagent Kit (Takara). RT-qPCR was performed on the QuantStudio™ 1 PLUS Real-Time PCR System (Thermo Scientific) using SYBR Green Master Mix (Sangon Biotech) to verify the expression profiles of the differentially expressed mRNAs (dif-mRNAs). The primers used were designed using Oligo 7 version 7.60 (OLIGO; Supplementary Table S1). All primers were synthesized by Sangon Biotech (Shanghai) Co. Relative dif-mRNA-expression levels were assessed using the 2^−ΔΔCt method, and the data were converted to log2-fold changes to enhance the clarity and facilitate interpretation of the data.

Statistical analysis

Experimental data were analysed to determine significant differences using Prism version 5.0 (GraphPad) and are expressed as the mean ± standard deviation. *p < 0.05 was considered significantly different, and **p < 0.01, ***p < 0.001, and ****p < 0.0001 were considered highly significantly different.

Results

PRRSV infected and damaged the testes, epididymides, and accessory sex glands of the male reproductive system in piglets

To investigate PRRSV infection in the testes of male piglets, 7-day-old male piglets were first subjected to nasal inoculation with PRRSV. Viremia was observed in the infected piglets starting at 1 dpc. Fever was observed in the infected piglets starting at 2 dpc, followed by respiratory symptoms such as shortness of breath, coughing, and sneezing at 3 dpc. Additionally, reproductive disorders, including testicular fever (10/10), mild redness (4/10), and sensitivity to touch (3/10), were observed in the infected group throughout the experiment.

All piglets survived the experiment. Infected and control-treated piglets were euthanized at 7 and 30 dpc, respectively. The intact male reproductive systems of the piglets were isolated (Figure 1a). Necropsies revealed pronounced inflammatory reactions in the male reproductive system of the piglets. Throughout the experiment, the infected piglets showed enlarged testes and epididymides with significant vascular congestion, in stark contrast with the mock-infected piglets. In addition, the prostate gland, seminal vesicles, and bulbourethral glands were visibly inflamed and swollen, and the ureters appeared red. At 30 dpc, the infected group exhibited thickening of the ureters.

Figure 1.

PRRSV pathogenicity in the reproductive system of male piglets. (a) Gross lesions observed in the reproductive system of male piglets at 7 and 30 dpc. (c) Histopathological changes in the testes, epididymides, bulbourethral glands, prostate gland, and seminal vesicles of male piglets (200×). Scale bars = 100 μm. (c) Changes in the diameter of the seminiferous tubules in each group. ****p < 0.0001; ***p < 0.001; **p < 0.01; *p < 0.05; ns, not significant. (d) Detection of viral-antigen distributions in the testes, epididymides, bulbourethral glands, prostate gland, and seminal vesicles via IHC staining. Scale bars = 50 μm. (e) Detection of viral loads in the blood from 1 to 30 dpc via RT-qPCR. (f) Detection of viral loads in the blood, testes, epididymides, bulbourethral glands, prostate gland, and seminal vesicles via RT-qPCR.

We conducted histopathological analysis to investigate the direct effects of PRRSV infection on the male genital tract (Figure 1b). Piglets in the infected groups exhibited impaired growth and development in their testes. At 7 dpc, the testes displayed a sparse arrangement, luminal atrophy, interstitium widening, and a disorganized and irregular arrangement of spermatogenic cells in the lumen. By 30 dpc, the testes showed fewer intraluminal spermatogenic cells, disordered maturation in degenerate germinal epithelial cells, and multinucleated germ cells sloughing into the tubular lumina. Furthermore, we observed a significant impact of PRRSV infection on the development of spermatogenic tubules, resulting in a reduction in their diameter (Figure 1c).

Infected piglets also displayed cell death in the epididymides, with necroptosis or apoptosis occurring in individual epithelial cells at 7 dpc and necrotic cellular residues at 30 dpc. The infected piglets exhibited growth and developmental disorders in their seminal vesicles and prostate glands at both time points. The infected piglets exhibited more irregular lumens, disorganized epithelial cells, and inconspicuous columnar morphology than the mock-infected groups. Dysplasia and glandular atrophy occurred in the bulbourethral glands of the infected piglets. At 7 dpc, the vesicles had sparse epithelial cells, but no other changes were evident. By 30 dpc, the bulbourethral-gland lobes had decreased in size, and the vesicles had begun to undergo atrophy. No histopathological abnormalities were observed in any of the control sections.

PRRSV colonization was investigated via RT-qPCR analysis. PRRSV was detected in blood and various parts of the male reproductive system, including the testes, epididymides, seminal vesicles, prostate gland, and bulbourethral glands. The presence of PRRSV antigen could be detected in the blood of infected piglets at 1 dpc, with the highest viral load observed at 9 dpc (Figure 1e). In the reproductive system the testes, epididymides, and seminal vesicles exhibited the highest viral loads. In contrast, PRRSV was undetectable in all examined tissues from the mock-infected groups throughout the experiment (Figure 1f).

IF staining revealed the presence of PRRSV in various reproductive organs, including the testes, epididymides, prostate gland, seminal vesicles, and bulbourethral glands (Figure 1d). PRRSV-positive IF signals were detected in Leydig cells, Sertoli cells, and spermatogonia in the testes. Furthermore, PRRSV-infected macrophages were observed in the seminiferous tubules. In the epididymides, PRRSV-positive cells were primarily located in the efferent ducts, and detached PRRSV-infected tall columnar cells were observed in the lumen. PRRSV-positive cells were also observed in the interstitium of the epididymides. The epithelium on the luminal surface of the prostate gland showed the strongest PRRSV-positive IF staining. Numerous epithelial secretory cells in the prostate gland were infected with PRRSV. The PRRSV-positive signals were mainly distributed in the mucosa of the seminal vesicle glands. However, follicles in the urethral bulbourethral glands showed no signs of PRRSV infection.

Identification and quantification of dif-mRNAs

mRNA-expression levels were determined via high-throughput sequencing of testicular tissues from piglets in the PRRSV-infected and control groups. Previous data revealed robust responses in the testes at 7 dpc during the acute phase of infection. Three testicular samples were collected from both the infected and control groups at 7 dpc, and RNA was extracted. Thus, six RNA samples were sequenced, resulting in a total of 121.74 Gb of data. After processing, the clean data for all samples exceeded 19.05 Gb, and the percentage of Q30 bases was ≥ 93.12%, indicating that high-quality data were obtained with all samples (Figure 2a). Principal component analysis (PCA) showed high intragroup reproducibility and significant intergroup differences, indicating the feasibility of further DEG analysis (Figure 2b,c). We identified 4627 DEGs, comprising 2203 upregulated mRNAs and 2424 downregulated mRNAs (Figure 2d). The results were consistent with the transcriptome-sequencing results. The resulting transcriptome data were deposited in the SRA databank (www.ncbi.nlm.nih.gov/sra) and are available under the accession number PRJNA1070966.

Figure 2.

Identification of mRNAs expressed in response to PRRSV infection in the testes of piglets at 7 dpc. (a) Statistical analysis of the mRNA-sequencing data. (b) Venn diagram illustrating the number of detected mRnas. (c) PCA based on fragments per kilobase of exon model per million mapped fragments of all dif-mRnas. The ellipses represent the confidence intervals for each group. (d) Volcano plots were used to identify significantly dif-mRNAs transcripts under different biological conditions. (e) Validation of the RNA-seq data via RT-qPCR analysis. The blue and red bars represent the RNA-seq results and RT-qPCR validation results, respectively. The RT-qPCR validation results are displayed as the mean log2 (fold change) ± standard deviation (error bars) from three independent experiments.

To confirm the accuracy of the transcriptomics data, eight randomly selected dif-RNAs (ALG8, ECH1, GRIK1, GAS2L3, NCF2, NRXN3, FAM171A2, and SPAG17) were validated via RT-qPCR method. The RT-qPCR results for these dif-RNAs were highly consistent with the sequencing results, indicating that significant differences in mRNA-expression levels occurred between the infected and mock-infected groups (Figure 2e).

PRRSV induced testicular-growth disorders, apoptosis, necroptosis, and dysregulated hormone secretion

Sertoli cells play crucial roles in establishing the BTB, and the period between 7 and 45 days of age is critical for Sertoli cell differentiation [26]. To examine the effect of PRRSV on the proliferative activity of Sertoli cells in the testes, we co-stained testis sections for the markers, SOX9 and PCNA (Figure 3a). SOX9 serves as a marker for all Sertoli cell stages, whereas PCNA indicates the proliferative status of Sertoli cells. IF staining revealed increased numbers of SOX9⁺ cells in the piglet testes in all groups from 7 to 30 dpc. However, the increase was smaller in the infected groups. Furthermore, significantly fewer SOX9⁺ cells were observed in the testes of infected piglets (p < 0.01). The number of SOX9⁺ PCNA⁺ cells and the proportion of proliferating SOX9⁺ cells generally decreased with age. Notably, the testes of piglets in the infected groups exhibited fewer SOX9⁺ PCNA⁺ cells than those in the mock-infected groups (p < 0.05; Figure 3b).

Figure 3.

PRRSV infection induced testicular growth and developmental disruption, apoptosis, and hormonal imbalances. (a) Immunostaining of porcine testis sections for SOX9 and PCNA expression. Scale bars = 50 μm. (b) Counts of SOX9⁺ and SOX9⁺ PCNA⁺ cells per cross-section of seminiferous cords/tubules in porcine testes are shown. (c) Immunostaining of porcine testis sections for β-catenin and ZO-1. Scale bars = 50 μm. (d) Detection of apoptosis in the testes by performing TUNEL assays. Scale bars = 50 μm. (e) ELISA analysis of serum T and AMH concentration. ****p < 0.0001; ***p < 0.001; **p < 0.01; *p < 0.05; ns, not significant. (f) Analysis of dif-mRNAs revealed enrichment for GO terms associated with testicular growth and development, apoptosis, and hormone secretion. The top 20 GO pathways are shown. (g) Analysis of dif-mRNAs revealed enrichment for KEGG pathways associated with testicular growth and development, apoptosis, and hormone secretion. The top 15 KEGG pathways are shown. The rich factor indicates the ratio of dif-mRNAs enriched within a specific pathway. The sizes and colours of the solid circles represent the number of enriched dif-mRNAs and the significance of the enrichment, respectively.

β-catenin and ZO-1 play crucial roles as major structural components of the BTB, which undergoes rapid development before sexual maturation, signifying the formation of the BTB [26]. β-catenin is a component of the testis-specific adherens junction in the testicular mesenchyme [27], whereas ZO-1 is a crucial element of the TJ responsible for segregating the spermatogonial epithelium into the basal and luminal chambers [28]. To evaluate BTB growth and development, we co-stained testis sections for ZO-1 and β-catenin (Figure 3c). ZO-1 and β-catenin levels increased significantly between 7 and 30 dpc in the testes of all piglets. However, the increase was significantly smaller in the infected groups. Additionally, we observed loosening, disintegration, and even the collapse of some TJs in piglets in the infected groups.

Apoptosis of early differentiated spermatogonia or Sertoli cells during BTB development can have serious consequences [29]. To investigate PRRSV-induced apoptosis in testicular cells, we conducted TUNEL experiments on testis sections (Figure 3d). Throughout the experiment, the numbers of apoptotic cells were significantly higher in the testes of the infected groups than in the mock-infected groups. The apoptotic cells were primarily located in the mesenchyme. Furthermore, significantly more Sertoli cells and spermatogonial nuclei underwent consolidation and cleavage, with positive TUNEL staining.

T is primarily secreted by interstitial cells in the testes and plays a key role in maintaining the integrity and function of the BTB [30]. AMH is a member of the transforming growth factor beta (TGF-β) family that is specifically expressed in immature Sertoli cells [31]. We observed that at 7, 14, and 21 dpc, the serum levels of T and AMH were significantly lower in the infected piglets than in the control piglets (p < 0.01; Figure 3e). However, at 28 dpc, the AMH levels were significantly higher in the infected piglets than in the mock-infected group (p < 0.01), whereas the T levels were not significantly different (p > 0.05).

After sequencing, we examined the functions of the dif-mRNAs using GO and KEGG pathway analyses. We screened 20 GO terms and 15 KEGG terms that were significantly associated with testicular growth and development, hormone secretion, apoptosis, and necroptosis (Figure 3f,g). In terms of GO terms associated with testicular growth and development, PRRSV infection caused enrichment for the following terms: seminiferous tubule development (GO: 0072520), reproductive structure development (GO: 0048608), Sertoli cell development (GO: 0060009), and mesenchymal cell differentiation (GO: 0048762). In addition, the GO terms positive regulation of necroptotic process (GO: 0060545), regulation of cell-cell adhesion (GO: 0022407), cellular developmental process (GO: 0048869), meiotic cell cycle process (GO: 1903046), regulation of cell cycle process (GO: 0010564), and developmental process involved in reproduction (GO: 0003006) were significantly reduced, suggesting that PRRSV infection may have impaired testicular growth and development.

Regarding GO terms associated with hormone secretion, PRRSV infection was negatively associated with the following terms: gonad development (GO: 0008406), regulation of hormone levels (GO: 0010817), response to hormone (GO: 0009725), male gonad development (GO: 0008584), cellular response to luteinizing hormone stimulus (GO: 0071373), and luteinizing hormone signalling pathway (GO: 0042700), suggesting that PRRSV infection may lead to poorer androgen secretion in the testes. PRRSV infection significantly upregulated the following GO terms: positive regulation of the apoptotic process (GO: 0043065), regulation of apoptotic process (GO: 0042981), positive regulation of the necroptotic process (GO: 0060545), and TRAIL binding (GO: 0045569). These data suggest that PRRSV infection may promote both necroptosis and apoptosis in testicular cells.

In terms of KEGG pathways associated with testicular growth and development, PRRSV infection mainly led to disruption of the following pathways: biosynthesis of unsaturated fatty acids (map01040), pantothenate and CoA biosynthesis (map00770), cell cycle (map04110), steroid biosynthesis (map00100), fat digestion and absorption (map04975), and glycolysis/gluconeogenesis (map00010), whereas adherens junction (map04520), and tight junction (map04530) were significantly downregulated, resulting in impaired testicular growth and development in infected piglets. Regarding KEGG pathways associated with hormone secretion, PRRSV infection significantly downregulated the following pathways: steroid hormone biosynthesis (map00140); cholesterol metabolism (map04979); steroid biosynthesis (map00100); and growth hormone synthesis, secretion, and action (map04935), resulting in disturbed testicular androgen secretion. In terms of KEGG pathways associated with cell death in testicular cells, PRRSV infection significantly enriched the following pathways: upregulation of necroptosis (map04217), cellular senescence (map04218), apoptosis (map04210), and autophagy – animal (map04140), indicating that PRRSV infection may lead to severe necroptosis and cell death.

PRRSV induced an intense inflammatory response in the testes

Generally, testicular inflammation results in tissue swelling and congestion, leading to an increased tissue weight [32]. At 7 and 30 dpc, the testicular TSIs in the infected groups were significantly higher than those in the mock-infected group (p < 0.01; Figure 4a).

Figure 4.

PRRSV infection induced testicular inflammatory responses. (a) TSI changes in each group. (b) Rectal and testicular temperature changes in each group from 1 to 30 dpc. (c) The expression levels of IFN-γ, TNF-α, IL-6, IL-1α, IL-1β, and IL-10 in the testes were measured via ELISA analysis. ****p < 0.0001; ***p < 0.001; **p < 0.01; *p < 0.05; ns, not significant. (d) IHC staining for IFN-γ in porcine testis tissue sections. Scale bars = 100 μm. (e) IHC staining for IL-6 in porcine testis tissue sections. Scale bars = 100 μm. (f) GO-enrichment analysis of dif-mRNAs associated with inflammation. The top 20 GO pathways are shown. (g) KEGG enrichment analysis of dif-mRNAs associated with inflammation. The top 15 KEGG pathways are shown. The sizes and colours of the solid circles represent the number of enriched dif-mRNAs and the significance of the enrichment, respectively.

The testes maintained a temperature that was slightly lower than the core body temperature of the piglets [33]. Abnormally high temperatures can induce heat stress. In the infected group, testicular temperatures increased at 2 dpc and remained above 35°C from 3 to 11 dpc. The increase in temperature was significantly greater than that observed in the mock-infected group (p < 0.0001). The highest average testicular temperature was 35.66°C at 6 dpc. By 23 dpc, the testicular temperatures of all infected piglets returned to normal, showing no significant differences compared with those in the mock-infected group. Additionally, the increased testicular temperatures in the infected piglets paralleled the rise in their rectal temperatures. From 1 to 22 dpc, the rectal temperature of piglets in the infected group was significantly higher than that of those in the mock-infected group. The highest recorded temperature was at 6 dpc, reaching 41.34 °C. Throughout the experiment, the rectal and testicular temperature of the control piglets remained within the normal range (Figure 4b).

Cytokines play crucial roles in inflammatory responses. To assess the impact of PRRSV infection on testicular inflammation, we continuously monitored changes in the testicular-cytokine profiles (Figure 4c). The results revealed significantly higher TNF-α, IL-1α, IL-1β, and IL-10 levels at both 7 dpc and 30 dpc in the infected groups than in the mock-infected groups (p<0.001). At 7 dpc, the infected group also showed significantly higher IL-6 levels than the mock-infected group at 7 dpc (p < 0.0001), although no significant difference was observed at 30 dpc (p > 0.05). In addition, no significant difference was observed in testicular IFN-γ levels between both groups at 7 and 30 dpc (p < 0.05). To gain insight into the cytokine-expression levels in testicular cells, we conducted IF-staining experiments for IFN-γ and IL-6 with testicular sections (Figure 4d,e). The testicular IL-6 levels in the infected groups were consistently higher than those in the mock-infected groups throughout the experiment. At 7 dpc, no significant difference was found in testicular IFN-γ expression between the infected and control groups. However, at 30 dpc, the infected group showed significantly greater testicular IFN-γ expression than did the mock-infected group. Notably, IL-6 was primarily distributed in the seminiferous tubules.

The transcriptome-sequencing results suggested that pathways involved in the inflammatory response were significantly affected by PRRSV infection. We screened 20 GO and 15 KEGG terms thought to participate in testicular inflammatory responses (Figure 4f,g). GO enrichment analysis revealed that PRRSV infection significantly upregulated the inflammatory response pathway (GO: 0006954). The most significantly enriched GO signalling pathways in the PRRSV-infected group (compared with those in the mock-infected group) were neutrophil chemotaxis (GO: 0030593), chemokine-mediated signalling pathway (GO: 0070098), and cytokine-mediated signalling pathway (GO: 0019221). PRRSV infection induced an intense storm of inflammatory factors, mainly by regulating cytokines, as reflected by significant upregulation of the following GO terms: cellular response to cytokine stimulus (GO: 0071345), response to cytokine (GO: 0034097), regulation of cytokine production (GO: 0001817), cytokine activity (GO: 0005125), cytokine receptor binding (GO: 0005126), and cytokine receptor activity (GO: 0004896). Several interesting signalling pathways that favour inflammation, such as chemokine receptor binding (GO: 0042379), CCR chemokine receptor binding (GO: 0048020), positive regulation of dendritic cell cytokine production (GO: 0002732), and chronic inflammatory response (GO: 0002544), were also regulated by PRRSV infection.

KEGG enrichment analysis revealed that PRRSV infection mediated inflammation mainly through the direct activation of inflammatory cytokine signalling pathways, such as viral protein interaction with cytokine and cytokine receptor (map04061), cytokine-cytokine receptor interaction (map04060), and IL-17 signalling pathway (map04657). PRRSV infection also promoted inflammation through several specific signalling pathways, including the NOD-like receptor signalling pathway (map04621), the TNF signalling pathway (map04668), PPAR signalling pathway (map03320), and the NOD-like receptor signalling pathway (map04621). In addition, PRRSV infection upregulated the JAK-STAT signalling pathway (map04630), Toll-like receptor signalling pathway (map04620), chemokine signalling pathway (map04062), and NF-kappa B signalling pathway (map04064), which are important for cellular signalling. Notably, PRRSV infection in the testes enriched several pathways that were upregulated in certain autoimmune diseases, including graft-versus-host disease (map05332), systemic lupus erythematosus (map05322), and coronavirus disease – COVID-19 (map05171). These findings suggest that PRRSV may trigger the immune system to erroneously attack the body, leading to inflammation and injury. In addition, PRRSV infection enriched the arachidonic acid metabolism (map00590) and thermogenesis (map04714) pathways, leading to inflammation by regulating metabolite levels.

PRRSV induced severe oxidative stress damage in the testes

To investigate the effects of PRRSV on oxidative stress in the testes, we measured the concentrations of H₂O₂, T-AOC, MDA, GSH, GSSG, SOD, CAT, and GSH-PX (Figure 5a). We found that PRRSV infection resulted in significant increases in the levels of the oxidative stress-inducing agents, MDA and H₂O₂, within the testes (p < 0.001). Conversely, the activities of the antioxidant enzymes, SOD, T-AOC, GSH-PX, and CAT, decreased significantly (p < 0.05) after infection.

Figure 5.

PRRSV infection induced oxidative-stress damage in the testes of male piglets. (a) Oxidation index and antioxidant capacity of the testes in male piglets. We measured the SOD, GSH-PX, T-AOC, and CAT activities in the testes, as well as the H₂O₂ and MDA contents. ****p < 0.0001; ***p < 0.001; **p < 0.01; *p < 0.05; ns, not significant. (b) GO enrichment analysis of dif-mRNAs associated with oxidative stress. The top 20 GO pathways are shown. (c) KEGG-enrichment analysis of dif-mRNAs associated with oxidative stress. The top 15 KEGG pathways are shown. The sizes and colours of the solid circles represent the number of enriched dif-mRNAs and the significance of the enrichment, respectively.

Our transcriptome-sequencing results also indicated that PRRSV infection caused significant oxidative stress damage in piglet testes. We analysed the functions of dif-mRNAs using GO and KEGG pathway analyses. We identified 20 GO and 15 KEGG terms that were significantly associated with oxidative stress (Figure 5b,c). GO enrichment analysis indicated that the modulation of metabolic processes was the most significant mechanism whereby PRRSV induces oxidative stress. The most enriched GO terms included lipid oxidation (GO: 0034440), reactive oxygen species metabolic processes (GO: 0072593), and epoxide metabolic processes (GO: 0097176). PRRSV infection also affected the state of oxidative stress in testicular tissues by significantly enhancing several molecular-function terms, including oxidoreductase activity, acting on the CH-OH group of donors, NAD or NADP as acceptor (GO: 0016616); oxidoreductase activity, acting on the CH-OH group of donors (GO: 0016614); oxidoreductase activity, acting on CH-OH group of donors (GO: 0016684); peroxisome (GO: 0005777); peptide-disulphide oxidoreductase activity (GO: 0015037); oxidoreductase activity, acting on the CH-CH group of donors, NAD or NADP as acceptor (GO: 0016628); and oxidoreductase activity, acting on a haem group of donors, oxygen as acceptor (GO: 0016676). Furthermore, PRRSV infection exacerbated oxidative stress by negatively regulating antioxidant activity (GO: 0016209). Notably, PRRSV also affected testicular cells in terms of the response to oxidative stress (GO: 0006979) and response to reactive oxygen species (GO: 0000302) biological processes, leading to an increased susceptibility of testicular cells to oxidative stimulants.

The results of the KEGG enrichment analysis indicated that PRRSV infection primarily led to tissue damage by causing metabolic disorders, which in turn resulted in the excessive production of oxidative metabolites in tissues. Enriched pathways related to lipid metabolism included fatty acid degradation (map00071), chemical carcinogenesis – reactive oxygen species (map05208), and glyoxylate and dicarboxylate metabolism (map00630). The pathways type I diabetes mellitus (map04940), pyruvate metabolism (map00620), citrate cycle (TCA cycle) (map00020), pantothenate and CoA biosynthesis (map00770), and ascorbate and aldarate metabolism (map00053) were also enriched. Enriched amino acid metabolism pathways included beta-alanine metabolism (map00410), glutathione metabolism (map00480), and arginine and proline metabolism (map00330). PRRSV infection was also associated with the KEGG pathways, peroxisome (map04146) and ascorbate and aldarate metabolism (map00053), thereby negatively affecting the antioxidant capacity of testicular tissues. In addition, PRRSV infection indirectly promoted oxidative stress through other pathways such as the metabolism of xenobiotics by cytochrome P450 (map00980), p53 signalling pathway (map04115) and the C-type lectin receptor signalling pathway (map04625).

Discussion

To elucidate the overall effect of PRRSV infection on the male reproductive system, we isolated and comprehensively analysed the male reproductive system in piglets. The presence of PRRSV in the testes, epididymides, and bulbourethral glands has been previously documented [23,24]. The results of this study further revealed that PRRSV could infect nearly all organs of the male reproductive system, including not only the testes, epididymides, and bulbourethral glands, but also the seminal vesicles and prostate gland. In addition, our current findings provide an updated explanation for PRRSV transmission via semen, which is one of the primary routes of PRRSV transmission. Semen consists of sperm and plasma. Some researchers have hypothesized that PRRSV is primarily transmitted through the semen after it infects spermatozoa in the testes [34,35]. However, our findings indicate that the source of PRRSV in semen may also encompass seminal plasma secreted by accessory glands, such as the prostate gland, seminal vesicles, and bulbourethral glands. Interestingly, in pathological and IHC sections, we also observed that PRRSV-infected macrophages crossed the BTB and directly infiltrated the seminiferous tubules. Macrophages are target cells of PRRSV [36]. In healthy adult boars, macrophages are only present in the testicular interstitium because of the BTB [27]. We hypothesize that this phenomenon reflects the immaturity of the BTB in piglets and that macrophages carrying PRRSV can pass directly through the BTB and infect Sertoli cells along with spermatogonia. This mechanism is likely to have more serious consequences in piglets than a PRRSV infection might in adult boars, potentially leading to impaired spermatogenesis or infertility in piglets after they reach sexual maturity.

Piglets up to 30 days old are most susceptible to PRRSV infection [37]. Irreversible damage to the BTB in boars early in life can lead to impaired fertility or lifelong infertility [17]. Our IHC results revealed that PRRSV was capable of infecting Sertoli cells, which form the BTB, as well as spermatogonia, which are shielded by the BTB. To better understand the complex mechanisms whereby PRRSV infection impaired the function of the BTB, we analysed testicular growth and development, apoptosis, necroptosis, and hormone secretion after PRRSV infection. To assess testicular development, we initially measured the diameter of the seminiferous tubules in HE-stained sections. Our findings indicate that PRRSV infection led to impaired growth and development of seminiferous tubules, implying that infection potentially inhibited the development of spermatogonia and Sertoli cells. TJs established between Sertoli cells serve as the structural foundation of the BTB. The period between 7 and 45 days of age is crucial for Sertoli cell proliferation and differentiation [26]. Damage may lead to BTB developmental disorders.

To investigate this phenomenon, we performed SOX9 and PCNA IF staining with Sertoli cells and found that PRRSV infection significantly reduced the abundance and proliferative activity of Sertoli cells. We also investigated the expression of β-catenin and ZO-1, the primary components of adherens junctions and TJs, respectively. β-catenin and ZO-1 are expressed rapidly before sexual maturation and generally exhibit a pattern of conjugate staining at 130 days, indicating the formation of the BTB [38]. Our findings suggest that PRRSV infection led to substantial and lasting decreases in β-catenin and ZO-1 expression in the testes of piglets, resulting in severe growth and developmental impairment. TUNEL experiments further demonstrated that PRRSV induced apoptosis in developing Sertoli cells and spermatogonial cells, which explains the aforementioned results.

Hormone secretion plays crucial roles in regulating testicular development [31]. T is an important androgen secreted by the testes that plays key roles in regulating the maturation and growth of reproductive organs [39]. T also participates in the reorganization of cellular connections in the BTB [40]. In this study, we observed a significant reduction in T cell secretion following PRRSV infection. AMH, a member of the TGF-β family, was expressed at particularly high levels in immature Sertoli cells [31]. AMH inhibits male Müllerian duct development and plays roles in testicular differentiation and development [41]. We found that PRRSV infection significantly decreased AMH levels in male piglets, indicative of impaired Sertoli cell function, which could further impede testicular development.

To gain a deeper understanding of the mechanism whereby PRRSV might induce inflammation, we examined cytokine levels in the testes. We observed significant upregulation of TNF-α, IL-1α, IL-1β, IL-6, and IL-10 in the testes, leading to a cytokine storm. Furthermore, we performed IF staining for IL-6, TNF-α, and IFN-γ. Staining for IL-6 revealed that it was primarily localized in the seminiferous tubules, indicating that an intense inflammatory response occurred. IL-6 can disrupt the BTB by inhibiting protein degradation or activating extracellular signal-regulated kinase ERK via phosphorylation in Sertoli cells [42]. In contrast, TNF-α showed widespread distribution in the testes. TNF-α can directly downregulate occludin, ZO-1, and N-cadherin expression, thereby affecting the integrity of the BTB [43]. Although IFN-γ is an important activator of macrophages and plays crucial roles in antiviral responses [43], we did not detect significant IFN-γ upregulation at 7 dpc. This finding could be attributed to the ability of PRRSV to escape the immune response by suppressing IFN-γ secretion. This mechanism, widely observed in the respiratory tract, appears to have a similar effect in the testes [44].

Furthermore, our transcriptome-sequencing results validated our hypotheses that PRRSV infection in piglets impairs BTB development. GO analysis revealed that PRRSV infection primarily induced inflammation by regulating numerous cytokine-related genes, resulting in inflammatory damage in the testes. KEGG analysis revealed that PRRSV primarily induced inflammation by directly activating inflammatory cytokine-signalling pathways. Additionally, several specific signalling pathways, such as the NOD-like receptor, TNF-, PPAR-, and JAK-STAT-signalling pathways, help regulate inflammatory responses. Importantly, PRRSV shares similar signalling pathways with autoimmune diseases such as graft-versus-host disease, systemic lupus erythematosus, and coronavirus disease 2019. These findings suggest that PRRSV may elicit a similar pathogenic mechanism, leading to testis inflammation by causing the immune system to initiate an autoimmune response [45–47]. Moreover, PRRSV can stimulate the production of inflammatory mediators such as arachidonic acid, which can induce fever, pain, increased vascular permeability, and leukocyte exudation [48].

Oxidative stress is a prevalent factor in testicular dysfunction that can lead to impaired BTB damage, ultimately resulting in testicular apoptosis [49]. This phenomenon occurs when the production of free oxygen radicals exceeds the antioxidant capacity of a tissue. Our results indicated that PRRSV infection increased the levels of MDA and H₂O₂ while suppressing the functions of SOD, T-AOC, GSH-PX, and CAT in the testes.

Transcriptome analysis revealed that PRRSV induced peroxidation in the testes by upregulating the expression of oxidation-related genes and downregulating antioxidant enzyme-related genes. Such outcomes can exacerbate damage caused by peroxidation products by altering the response of the testes to oxidative stress. Additionally, PRRSV-induced oxidative stress damage is caused by the robust production of peroxidation products, owing to disturbed substance metabolism in testicular tissues and reduced antioxidant-enzyme activity through various pathways. Interestingly, we also identified activation of the cytochrome P450 pathway (map00980), the p53 signalling pathway (map04115), and the C-type lectin receptor signalling pathways (map04625). Cytochrome P450 is linked to mitochondrial oxidative stress and produces N-acetylbenzoquinone imine, a compound with potent oxidative properties that can result in mitochondrial dysfunction [50]. p53 can activate cellular necrotic pathways and induce reactive oxygen species production during oxidative stress [51].

In conclusion, we systematically analysed the consequences of PRRSV infection in the male reproductive system of piglets. PRRSV infected multiple organs of the male reproductive system, including the testes, epididymides, seminal vesicles, prostate gland, and bulbourethral glands. Furthermore, PRRSV-infected macrophages were present in the seminiferous tubules of piglets, and infected Sertoli cells were present in seminiferous tubules and spermatogonia. These results offer new insights into the effects of PRRSV infection on the BTB in male piglets. PRRSV infection significantly impaired testicular development and function through a multifactorial mechanism. This mechanism involves the induction of cell death via apoptosis or necroptosis, dysregulation of hormones, initiation of intense inflammation, and generation of severe oxidative stress. Overall, our findings revealed the harmful effects of PRRSV infection on the male reproductive system and provide important insights into the intricate and interconnected factors that contribute to testicular dysfunction.

Funding Statement

This work was supported by the Key R&D Projects of the Sichuan Science and Technology Program [No. 2023YFN0021], the Basic construction task of experimental pig resource development and innovation team [NCTIP-XD1C14], the Innovation and Application of Key Technologies of Natural Medicine to Improve the Breeding Rate of Conservation Pigs [NCTIP-MY23006], Key Research and Development Project of Sichuan Science and Technology Plan [Grant No. 2022YFN0007], the Porcine Major Science and Technology Project of Sichuan Science and Technology Plan [No. 2021ZDZX0010-3], the Chongqing Municipal Technology Innovation and Application Development Project [No. cstc2021jscx-dxwt BX0007], Key Research and Development Program in rural areas of Sichuan Science and Technology Department [Grant No. 2020YFN0147], Agricultural Industrial Technology System of Sichuan Provincial Department of Agriculture [Grant No. CARS-SVDIP].

Disclosure statement

No potential conflict of interest was reported by the author(s).

Author contributions

Huang Bingzhou: Conceptualization, Methodology, Investigation, Formal analysis, Writing – Original Draft, and Visualization. Li Fengqin: Conceptualization, Methodology, Investigation, and Writing – Original Draft. You Dong: Methodology, Investigation, and Writing – Original Draft. Deng Lishuang: Methodology, Formal analysis, and Writing – Original Draft. Xu Tong: Conceptualization, Methodology, Writing – Review & Editing, Investigation, and Visualization. Lai Siyuan: Project administration, Data Curation, Investigation, Writing – Original Draft, and Validation. Ai Yanru: Project administration, Data Curation, Investigation, Writing – Original Draft, and Validation. Huang Jianbo: Methodology, Investigation, Writing – Review & Editing, and Investigation. Zhou Yuancheng: Resources, Investigation, Investigation, Writing – Review & Editing, and Supervision. Ge Liangpeng: Resources, Project administration, Writing – Review & Editing, and Supervision. Zeng Xiu: Resources, Project administration, Writing – Review & Editing, and Supervision. Xu Zhiwen: Conceptualization, Methodology, Project administration, Writing – Review & Editing, and Supervision. Zhu Ling: Conceptualization, Methodology, Project administration, Writing – Review & Editing, and Supervision.

Data availability statement

Raw data generated in this study were deposited in the Sequence Read Archive Database (www.ncbi.nlm.nih.gov/sra) under accession number PRJNA1070966 and Zenodo (zenodo.org) under accession number 11,082,324.

Supplemental data

Supplemental data for this article can be accessed online at https://doi.org/10.1080/21505594.2024.2384564