Cryopreservation stands as the most efficacious method for the long-term preservation of mammalian sperm [1]. The principal advantage of cryopreserving boar sperm is the storage of genetic material in germplasm banks and the facilitation of long-distance transportation of sperm [2,3]. However, the utilization of frozen boar semen in artificial insemination is extremely limited, with the majority resorting to fresh semen [4]. The primary reason for this is that the freeze-thawing procedures markedly impair sperm function and viability, thereby reducing reproductive performance [5]. Notably, not all sperm cells sustain the same level of damage [4], and each ejaculate exhibits distinct cryotolerance, with evident variations between individuals and among ejaculates from the same individual [6]. Consequently, considerable endeavors have been dedicated to identifying freezability markers that assist in predicting the freezing tolerance of semen ejaculates.

Omics techniques have emerged as powerful tools for unraveling molecular regulatory networks and mining biological genetic markers, thereby providing a valuable and broad strategy for studying these multifactorial processes [7]. Currently, the cryopreservation of boar semen has been extensively studied, including comparative analyses of genomic [8] transcriptomic [9], proteomic [10], and epitranscriptomic [11] profiles between poor freezability ejaculates (PFE) and good freezability ejaculates (GFE). These subtle changes may ultimately be reflected in metabolomics studies. Metabolomics, which involves the identification and quantification of the metabolome, is broadly acknowledged as the omics discipline most closely linked to the phenotype [12,13]. Studies have demonstrated that sperm metabolites may directly or indirectly regulate signaling pathways related to sperm motility, hyperactivation, and energy acquisition [14]. Therefore, the selection of metabolomics to identify cryopreservation differential markers between GFE and PFE could offer valuable insights into the physiological and molecular mechanisms underlying sperm cryopreservation.

In recent years, metabolomics has been extensively employed in animal husbandry to identify differential markers of sperm and seminal plasma cryopreservation in boars [15], rams [16], bulls [17], and roosters [18]. This has contributed to enhancing our understanding of semen cryopreservation. During the freezing process, the seminal plasma is typically discarded, leaving the majority of sperm. Hence, identifying differentially expressed metabolites (DEMs) in sperm is crucial for understanding the reasons behind the variability in freezability among individuals after freezing. Metabolomic studies on boar sperm have revealed that the DEMs are predominantly enriched in pathways associated with sperm quality. Twenty-one DEMs in boar sperm were identified between the PFE and GFE groups of Chinese native pig [19]. Fifteen lipids showed differences between the GFE and PFE from Yorkshire boars [20]. However, significant differences in semen quality after cryopreservation are also observed among different breeds [21]. The availability of DEMs identified by previous studies to other breeds remains to be further determined. A comprehensive analysis encompassing a larger sample population from multiple breeds can provide more extensive information on metabolism, capture metabolic differences between breeds, and uncover more general and stable markers of boar sperm freezing differences. This provides a theoretical basis for the improvement of cryopreservation techniques for pig semen, which could help enhance the application value of cryopreserved pig semen.

This study aims to compare the metabolomic profiles of boar semen samples from the GFE and PFE groups across three breeds (Duroc, Landrace, and Large White boars), with the purpose of elucidating the metabolic pathways and key metabolites that might contribute to the observed differences between two groups. The results of this study would provide potential markers for assessing the freezing tolerance in boar sperm and offer a potential insight into the molecular mechanisms associated with differences in boar sperm cryopreservation capacity.