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After the sperm cryopreservation process, there frequently is a marked decrease in sperm motility, however, this loss of motility could be avoided or reduced by modifying the thawing extender. The aim of conducting this study was to evaluate treatments resulting in differences in pH of the thawing extender on post-thawing sperm quality, especially motility. The associated effects were evaluated of the thawing extender pH when there were pH values of 7, 8 and 9 on values for sperm viability, acrosome integrity, motility and kinematic variables. To evaluate pH effects of the thawing extender on sperm motility, the procedures for evaluating the sperm variables were conducted with or without inclusion of caffeine in the extender. In addition, effects of thawing extender pH on the sperm subpopulations were analyzed. The results indicated that by increasing the pH of thawing extender to 9, sperm viability and acrosome status were not affected, and motility was markedly greater with there being similar values when there was a pH of greater alkalinity and pH 7 when caffeine was included for conducting the evaluations. Furthermore, for values of kinematic variables and sperm subpopulations, when there was a pH of 8 of thawing extender, sperm motility was maintained to a greater extent as storage duration advanced and the proportions of different sperm populations were also maintained during storage. In conclusion, when there was use of the thawing extender at pH 8, there was enhanced post-thawing sperm motility and preservation of motility as storage time advanced., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Simple Summary: It remains unclear whether the prolonged preservation of cells, such as sperm, in liquid nitrogen (LN2) leads to their deterioration over time. In this study, we investigated whether the conservation of Iberian pig semen for ten years results in a decline in quality and whether the evaporation of LN2 from the tanks between refills could be a contributing factor. Furthermore, the only way to improve already-frozen samples is to implement specific thawing protocols. Thus, our findings suggest that only samples consistently submerged in LN2 benefit the most from the application of thawing protocols, such as rapid thawing at 70 °C for 8 s. While partial conservation in LN2 and LN2 vapors does not result in a loss of quality over time, it does cause damage that thawing protocols cannot mitigate. This finding suggests that, independently of the lack of the effect of the level of storage in the tank, careful handling and optimization of thawing protocols are essential for the quality preservation of the cryopreserved sperm samples. 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. [ABSTRACT FROM AUTHOR]