Evidence of breed-dependent Aquaporin 3 expression associated with sperm cryotolerance in bulls

This study examines whether differences in genetic selection pressure between breeds are reflected in sperm cryoresistance and AQP3 abundance and dynamics in membrane domains. Semen from nine bulls (five Holstein and four Pirenaica, a minimally selected native breed) was analyzed under chilled and frozen-thawed conditions using 36 ejaculates (four per bull), each evaluated in both conditions. Sperm motility was analyzed using a CASA system, sperm viability was assessed by fluorescence with the SQS2 Seminal Quality System, and membrane and acrosomal integrity were analyzed by fluorescence microscopy. Spermatozoa were examined for the presence, abundance and distribution of AQP3 by Western blot and immunocytofluorescence (ICF). In chilled semen, Holstein exhibited a higher sperm viability, and membrane and acrosome integrity (p < 0.05). In frozen-thawed semen, Holstein bulls exhibited superior progressive motility (p < 0.05) and a higher proportion of sperm with intact plasma membranes and acrosomes (p = 0.05). The cryoresistance index was higher in Holstein samples for total motile sperm (p < 0.05). No significant differences in isocitrate dehydrogenase (IDH) activity were observed between breeds under either condition. ICF revealed that AQP3 was localized in the midpiece and principal piece. The freeze-thaw process led to a significant increase (p < 0.05) in the proportion of spermatozoa exhibiting AQP3 in the midpiece and principal piece of Holstein samples. Holstein bulls exhibited significantly higher AQP3 abundance than Pirenaica in frozen-thawed semen (p < 0.05). A similar tendency was observed in chilled samples, where Holstein semen showed higher AQP3 levels than Pirenaica (p = 0.09). The results suggest that higher sperm cryoresistance in Holstein bulls was associated with higher AQP3 abundance and capacity for AQP3 redistribution along the flagellum during cryopreservation.