Post-translational modifications (PTMs) are covalent modifications that alter protein folding, localization, activity, and interactions with other proteins [1]. By introducing diverse chemical groups, PTMs remodel chromatin architecture and regulate key biological processes, including cellular metabolism, protein degradation, and DNA replication [2]. To date, more than 400 distinct PTM types have been identified in both prokaryotic and eukaryotic systems, such as acetylation, phosphorylation, crotonylation, methylation, glycosylation, and ubiquitination [3]. PTMs frequently target the ε-amino group of lysine residues [4]. Notably, aberrant PTMs are implicated in the pathogenesis of neurodegenerative diseases, atopic dermatitis, cancer, and diabetes [5], [6], [7]. Moreover, PTMs are not restricted to histones but are also widely distributed across non-histone proteins, where they play critical roles in diverse cellular functions [8].

In eukaryotes such as yeast and humans, lysine crotonylation (Kcr) is an evolutionarily conserved acylation modification that is abundantly enriched at promoter and enhancer regions of transcriptionally active genes [9]. Kcr specifically marks active X- and Y-linked genes in post-meiotic male germ cells and regulates spermatogenesis, gene transcription, protein biosynthesis, folding, and degradation in males [10], [11], [12]. Lysine acetylation (Kac) is widespread in both prokaryotic and eukaryotic systems and serves as a hallmark of transcriptionally active chromatin. Kac is intimately linked to diverse cellular processes, including glycolysis, pyruvate metabolism, the tricarboxylic acid cycle, cellular development, and bacterial pathogenicity [13], [14], [15]. This modification is catalyzed by histone acetyltransferases (HATs) and removed by histone deacetylases (HDACs), regulating the dynamic acetylation of lysine residues [16].

The testes are responsible for spermatogenesis and androgen secretion [17], and their development directly determines the reproductive efficiency and breeding potential of pigs. Age is a key factor influencing male fertility. For Landrace pigs, puberty begins at around 5 months, with the production of morphologically mature sperm, and sexual maturity is reached by 7 to 9 months of age [18]. To date, numerous studies have performed transcriptomic and proteomic analyses of testicular tissues from pigs at different ages, identifying several key candidate genes and proteins, including PIWIL4, SPATA3, SPATA4, Aconitate hydratase (ACO2), Carboxypeptidase O (CPO), and 40S ribosomal protein S5 (RPS5) [19], [20], [21]. However, studies on PTMs of testicular proteins remain very limited. Here, we analyzed protein crotonylation and acetylation in the testes of 5- and 24-month-old pigs, corresponding to two age-associated stages of pubertal development and adulthood, with a focus on these two key stages rather than constructing a continuous developmental atlas. This study provided a comprehensive analysis of Kcr and Kac during testicular development and identified potential key modified proteins, providing new insights into the regulatory roles of PTMs in spermatogenesis in pigs and other farm animals.