RESUMEN
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.
RESUMEN
Sperm capacitation is a crucial step towards the acquisition of fertilizing capacity. Despite the attempts to mimic the in vivo situation, there is still a lack of standardization in vitro techniques. Bicarbonate and serum albumin (BSA) are routinely used, although controversial results are reported regarding the optimal concentration of each compound. In addition, whether caffeine is needed on in vitro capacitation media in boar sperm remains to be elucidated. Here, 18 boar commercial artificial insemination doses were used to test different concentrations of bicarbonate (19, 37 or 56 mM) in experiment 1, BSA (1.5, 3, 4.5 mg/mL) in experiment 2 and the presence or absence of caffeine (5.15 mM) experiment 3. We analysed at 0, 30 and 120 min of incubation at 38.5°C, 5% CO2 : Total motility (TMOT), membrane integrity (VIAB), acrosomal exocytosis (rAcro; H33342/PI/PNA), capacitation status (chlortetracycline staining CTC) and mitochondrial membrane potential (JC-1). The higher concentrations of bicarbonate (37 and 56 mM) decreased TM and VIAB (p < .01) but increased rAcro (p < .01) after 120 min of incubation compared to the fresh control. In contrast, only the BSA concentration of 3 mg/mL reduced the VIAB at 120 min, but all the concentrations tested increased the average of JC-1 and decreased TM (p < .01) throughout incubation compared to the fresh control. Finally, in experiment 3, when boar sperm were incubated in the capacitating media with bicarbonate, BSA and with or without caffeine, the capacitated pattern measured by the CTC technique and rAcro increased after 120 min of incubation (p < .01) compared to fresh control, either in the presence or in the absence of caffeine. In summary, our results suggested that the combination of capacitating components, like bicarbonate and BSA, contributed to increasing the proportion of capacitated boar spermatozoa, mitochondrial membrane potential as well as acrosomal exocytosis. However, caffeine did not significantly influence in vitro sperm capacitation in this species.