The conservation of genetic resources in pig breeds, notably the Iberian pig, is crucial for genetic improvement and sustainable production. Prolonged storage in liquid nitrogen (LN₂) is recognized for preserving genetic diversity, but potential adverse effects on seminal quality remain debated. This study aims to assess the impact of ten years of storage at different LN₂ levels and to optimize thawing protocols for Iberian pig sperm. Sperm samples from 53 boars were cryopreserved and stored at varying LN₂ levels and, a decade later, the samples were thawed at 37 °C for 20 s or at 70 °C for 8 s. Sperm motility, membrane integrity, acrosome status, and DNA fragmentation were evaluated in year 0 and year 10. Overall, no significant differences were observed in post-thaw sperm quality between storage levels in year 0 or year 10. But thawing at 70 °C 8 s showed significant improvements, particularly in samples that were always stored in LN₂, in all analyzed parameters except fragmentation, which was not affected by cryostorage. This study suggests that the long-term preservation of Iberian pig sperm does not affect quality over time, regardless of whether the samples were fully submerged in LN₂. Furthermore, it is determined that thawing at 70 °C for 8 s maximizes post-thaw sperm quality, especially in those samples stored constantly submerged in LN₂.

Subfertile boars often go undetected until they cause significant reproductive losses. Current semen quality assessments are limited in their ability to predict fertility, highlighting the need for complementary biomarkers. This study explored whether semen freezability could serve as an indirect indicator of boar fertility. Eighteen boars were classified based on historical fertility records and semen freezability, assessed by post-thaw quality. Fresh and post-thaw semen samples were analyzed using the CASA system and fluorescence microscopy. High-fertility boars showed significantly better motility and functional sperm parameters in fresh semen compared to low-fertility boars. However, these differences were mostly lost after cryopreservation. Conversely, boars with good freezability had consistently better post-thaw semen quality, though this did not correlate directly with higher fertility outcomes. Notably, a combined analysis revealed that boars with both high fertility and poor freezability had the lowest post-thaw semen quality. This suggests that cryopreservation may expose hidden sperm defects not detectable in fresh semen. Total motility was the only parameter associated with both fertility and freezability. In conclusion, while freezability alone may not directly predict fertility, it may help identify low-performing males. The combined assessment of fresh semen motility and freezability could support more effective boar selection strategies.

In vitro capacitation of boar spermatozoa remains highly variable among laboratories due to differences in media composition, incubation conditions, and previous semen handling. Since capacitation is a dynamic process, identifying how different media modulate specific sperm responses over time is crucial to improving the standardization of porcine in vitro fertilization protocols. This study evaluated the temporal dynamics of membrane lipid disorder, acrosomal integrity, mitochondrial activity, and motility patterns in response to different media. Commercial boar semen doses were centrifuged and resuspended in a non-capacitating control solution or in three capacitation media differing mainly in bicarbonate, caffeine, and energy substrate composition, and evaluated every 15 min using flow cytometry and computer-assisted semen analysis at 38 °C for up to 2 h. Media containing higher bicarbonate and caffeine concentrations induced rapid membrane lipid destabilization and increased acrosomal exocytosis during the initial incubation period. Subsequently, these changes were accompanied by a more rapid decline in cell viability over time. In contrast, the medium containing lower bicarbonate together with lactate and pyruvate preserved higher viability, mitochondrial activity, and a more sustained, linear movement pattern (higher linearity and straightness). Interestingly, training-associated responses were detected immediately at time 0, suggesting that semen storage conditions may prime spermatozoa before incubation. Overall, medium composition and pre-treatment handling conditions should both be considered when standardizing porcine reproductive technologies.

In internally fertilizing animals, such as the pig, males transfer not only spermatozoa but also a complex mixture collectively termed seminal plasma (SP). SP contains diverse bioactive components, including proteins, peptides, cytokines, and various RNA species. Growing evidence indicates that SP contributes not only to the sperm quality but also modulates the female’s immune responses. Major progress in biological knowledge and in the discovery of sperm quality biomarkers has been driven by advances in biotechnology and by the increasing affordability of omics technologies. With modern proteomic, (epi)genomic, transcriptomic, metabolomic, and functional analyses applied directly to semen, it is now possible to evaluate not only the functional state of the testes, epididymis, and accessory glands, but also to predict fertility through emerging biomarkers. Beyond diagnostics and fertility forecasting, new opportunities arise from the sperm quality biomarker definition using semen additives—such as specific molecules (proteins, peptides, enzymes) or extracellular vesicles (EVs). These EVs contain bioactive cargo protected within nanovesicles that can be isolated, stored, or even produced de novo in vitro. Boar spermatozoa are notoriously difficult to cryopreserve because they lose cholesterol during handling. EVs could fuse with sperm cells, deliver cholesterol‑rich lipids to the sperm membrane, and transfer specific proteins and nucleic acids. This raises the possibility that harvesting EVs, especially from highly fertile males, could be used in the future to improve sperm storage and even cryopreservation. Taken together, this review aims to list sperm fertility biomarkers that could be useful for the AI companies to discriminate sub-fertile males at an early stage.