PRRSV is a major pathogen that significantly impacts the swine industry worldwide, causing extensive economic losses and posing a serious threat to industry sustainability [1]. Primarily affecting the reproductive and respiratory systems, PRRSV infections lead to reproductive disorders in sows and respiratory distress in piglets [2]. In recent years, increased attention has been given to the impact of PRRSV on the male reproductive system [3]. Our previous study [4] demonstrated that, following PRRSV infection, the virus can persistently replicate in the reproductive tissues of boars, including the testes, epididymis, and vas deferens. This continuous viral replication leads to detrimental effects on semen quality, such as decreased sperm motility, increased morphological abnormalities, and reduced concentration [5]. Consequently, PRRSV infection significantly impairs semen quality [6]. This issue not only reduces the breeding value of infected boars but also increases the risk of virus transmission to sows through contaminated semen, which may compromise offspring health and cause substantial economic losses for swine operations [7].

The boar testis contains essential cells for reproductive health, including spermatogonia and Sertoli cells [8]. Spermatogonia, as stem cells, generate spermatozoa through mitosis and meiosis [9], while Sertoli cells provide structural and nutritional support, which is critical for spermatogenesis and involves the secretion of essential regulatory factors [10]. Additionally, Sertoli cells establish the blood-testis barrier (BTB) through tight junctions, thereby protecting developing germ cells from immune system attacks [11]. The proper function of these cell types is indispensable for maintaining male fertility. However, our previous study [4] indicate that PRRSV infection disrupts the testicular microenvironment, potentially leading to apoptosis or reduced proliferative activity in both spermatogonia and Sertoli cells, which may result in reproductive abnormalities in boars. Understanding PRRSV’s effect on these cell types, along with its influence on their regulatory pathways, is crucial to elucidate PRRSV's pathogenic mechanisms and develop effective treatments.

Non-coding RNAs (ncRNAs), particularly those involved in the competitive endogenous RNA (ceRNA) network, are central regulators of testicular function and male fertility. The ceRNA network is composed of various ncRNAs, including circular RNAs (circRNAs) and microRNAs (miRNAs), which interact to modulate gene expression and maintain cellular homeostasis. CircRNAs are a distinct class of ncRNAs characterized by their stable, covalently closed circular structures, which make them resistant to ribonuclease-mediated degradation [12]. CircRNAs are highly expressed in the testis and play pivotal roles in germ cell differentiation, sperm production, and regulation of testis-specific genes.[13], [14]. Acting as molecular sponges, circRNAs bind to miRNAs, thereby preventing miRNAs from interacting with their target mRNAs and modulating gene expression [15]. This regulatory mechanism within the ceRNA network significantly influences testicular development and function, and dysregulation of circRNAs may impair spermatogenesis, ultimately leading to infertility [16], [17], [18]. MiRNAs are another key class of small endogenous ncRNAs, regulate gene expression post-transcriptionally by binding to the 3' untranslated region of target genes, leading to mRNA degradation or inhibition of translation [19]. They play crucial roles within the ceRNA network by regulating essential cellular processes such as the cell cycle, apoptosis, differentiation, and stress responses [20].

Growing evidence indicates that virus-induced testicular dysfunction and developmental abnormalities are closely linked to the regulatory functions of ncRNAs, primarily through apoptosis of spermatogonia, Sertoli cells, and Leydig cells [4]. Several studies have identified potential ncRNA target genes and highlighted key regulators involved in testicular function. Among these, miR-34c stands out for its testis-specific expression and critical roles in germ stem cell maintenance and spermatogenesis. It has been shown to regulate NANOS2, essential for sperm production [21], and RAD54L, which is involved in DNA repair and homologous recombination [23]—processes vital for chromosomal stability and germ cell viability [22], processes necessary for chromosomal stability and germ cell survival . These findings suggest a broader significance of miR-34c in male reproductive biology.

This study aimed to investigate the effects of PRRSV on circRNAs and miRNAs within the ceRNA network in the testis, to elucidate the regulatory mechanisms of circ-107191-miR-34c-RAD54L in testicular development, germ cell integrity, and reproductive health. Our results provide insights into the mechanisms by which PRRSV impairs male reproductive function and suggest potential therapeutic targets for mitigating these effects in boars.