Cryopreservation is the most suitable method to preserve boar spermatozoa over long-term storage. However, freeze-thawing protocols inflict extensive damage to sperm cells, reducing their viability and compromising their fertilizing ability. In addition, high individual variability is known to exist between boar ejaculates, which may be classified as of good (GFE) or poor (PFE) freezability. While conventional spermiogram parameters fail to predict sperm cryotolerance in fresh spermatozoa, high levels of certain proteins, also known as freezability markers, have been found to be related to the sperm resilience to withstand freeze-thawing procedures. In this context, the hypothesis of this study was that aquaporins AQP3, AQP7, and AQP11 could be linked to boar sperm cryotolerance. Twenty-nine ejaculates were evaluated and subsequently classified as GFE or PFE based upon their sperm viability and motility at post-thawing. Fourteen ejaculates resulted to be GFE, whereas the other fifteen were found to be PFE. Relative abundances of AQP3, AQP7, and AQP11 and their localization patterns were evaluated in all fresh and frozen-thawed ejaculates through immunoblotting and immunocytochemistry. Prior to cryopreservation, relative amounts of AQP3 and AQP7 were found to be significantly (p < 0.05) higher in GFE than in PFE. In contrast, no significant differences (p > 0.05) between freezability groups were found for AQP11, despite GFE tending to present higher levels of this protein. The localization of AQP7, but not that of AQP3 or AQP11, was observed to be affected by cryopreservation procedures. In conclusion, these results suggest that AQP3 and AQP7 are related to boar sperm cryotolerance and may be used as freezability markers.

Contents Aquaporins (AQPs) play a vital role for the transport of water and solutes across cell membranes. Classification of these ubiquitous proteins into three categories (orthodox AQPs, aquaglyceroporins and superaquaporins) is based on their sequence similarity and substrate selectivity. In the male reproductive tract of mammals, most AQPs (except AQP6 and AQP12) are found in different organs (including testis, efferent ducts and epididymis). AQP1 and AQP9 are the most abundant AQPs in the efferent ducts and epididymis and play a crucial role for the secretion/reabsorption dynamics of luminal fluid during sperm transport and maturation. AQP3, AQP7, AQP8 and AQP11 are the most abundant AQPs in sperm and are involved in the regulation of their volume, which is required for the differentiation of spermatids into spermatozoa during spermatogenesis, as well as in sperm transit along environments of different osmolality (male and female reproductive tracts). While different studies conducted in oocytes and embryos have demonstrated that AQPs are important for cryotolerance, data in sperm are scarce. At present, mounting evidence indicates that AQP3, AQP7 and AQP11 are involved in the sperm response to variations of osmolality and to freeze‐thawing procedures. All these studies contribute to understand the physiology of both male reproductive tract and sperm, and open up new research ventures on the improvement of sperm cryopreservation protocols.

The present study aimed to determine the localisation of aquaglyceroporins 3 (AQP3) and 7 (AQP7) in bull spermatozoa and their relationship with the sperm cell's resilience to withstand cryopreservation (i.e. cryotolerance). A total of 18 bull ejaculates were cryopreserved and their sperm quality analysed before and after freeze-thawing. The presence and localisation of AQP3 and AQP7 was determined through immunoblotting and immunocytochemistry. AQP3 was found in the mid-piece and AQP7 in the mid-piece and post-acrosomal region of bull spermatozoa. Immunoblotting showed specific signal bands at 30 and 60kDa for AQP3 and at 25kDa for AQP7. Neither the relative abundance of AQP3 and AQP7 nor their localisation patterns was altered by cryopreservation but individual differences between bull ejaculates were found in immunoblots. In order to determine whether these individual differences were related to sperm cryotolerance, bull ejaculates were classified as having good (GFE) or poor freezability (PFE) on the basis of their sperm quality after thawing. While the relative abundance of AQP3 before cryopreservation did not differ between ejaculates with GFE and PFE, the abundance of AQP7 was higher in GFE than in PFE ejaculates. This finding was further confirmed through principal component and linear regression analyses. In conclusion, the relative abundance of AQP7 in fresh semen may be used as a marker to predict bull sperm cryotolerance.

Aquaporins (AQPs) are integral membrane water channels that allow transport of water and small solutes across cell membranes. Although water permeability is known to play a critical role in mammalian cells, including spermatozoa, little is known about their localisation in boar spermatozoa. Two aquaporins, AQP7 and AQP11, in boar spermatozoa were identified by western blotting and localised through immunocytochemistry analyses. Western blot results showed that boar spermatozoa expressed AQP7 (25kDa) and AQP11 (50kDa). Immunocytochemistry analyses demonstrated that AQP7 was localised in the connecting piece of boar spermatozoa, while AQP11 was found in the head and mid-piece and diffuse labelling was also seen along the tail. Despite differences in AQP7 and AQP11 content between boar ejaculates, these differences were not found to be correlated with sperm quality in the case of AQP7. Conversely, AQP11 content showed a significant correlation (P<0.05) with sperm membrane integrity and fluidity and sperm motility. In conclusion, boar spermatozoa express AQP7 and AQP11, and the amounts of AQP11 but not those of AQP7 are correlated with sperm motility and membrane integrity.

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