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Background:Aquaporins (AQPs) are a family of transmembrane water channels that includes orthodox AQPs, aquaglyceroporins (GLPs) and superAQPs. AQP3, AQP7, AQP9 and AQP11 have been identified in boar sperm, and they are crucial for sperm maturation and osmoregulation. Water exchange is an important event incryopreservation, which is the most efficient method for long-term storage of sperm. However, the freeze-thaw process leads to sperm damage and a loss of fertilizing potential. Assuming that the quality of frozen-thawed sperm partially depends on the regulation of osmolality variations during this process, AQPs might play a crucial role in boar semen freezability. In this context, the aim of this study was to unravel the functional relevance of the different groups of AQPs for boar sperm cryotolerance through three different inhibitors. Results:Inhibition of different groups of AQPs was found to have different effects on boar sperm cryotolerance. Whereas the use of 1,3-propanediol (PDO), an inhibitor of orthodox AQPs and GLPs, decreased total motility (P<0.05), it increased post-thaw sperm viability, lowered membrane lipid disorder and increased mitochondrial membrane potential (MMP) (P<0.05). When acetazolamide (AC) was used as an inhibitor of orthodox AQPs, the effects on post-thaw sperm quality were restricted to a mild increase in MMP in the presence of the intermediate concentration at 30 min post-thaw and an increase in superoxide levels (P<0.05). Finally, the addition of phloretin (PHL), a GLP inhibitor, had detrimental effects on post-thaw total and progressive sperm motilities, viability and lipid membrane disorder (P<0.05). Conclusions: The effects of the different inhibitors suggest that GLPs rather than orthodox AQPs are relevant for boar sperm freezability. Moreover, the positive effect of PDO on sperm quality suggests a cryoprotective role for this molecule.