Trehalose and sucrose in chicken semen cryopreservation: effects on post-thaw sperm quality and molecular...

Cryopreservation is essential for avian genetic resource conservation but often compromises post-thaw sperm quality. This study evaluated the effects of trehalose and sucrose as non-permeating cryoprotectants on chicken semen cryopreserved using a straw-based protocol with reduced dimethylformamide (DMF). Pooled semen from Green-legged Partridge roosters was assigned to a cryopreserved control group containing 6% DMF or to treatment groups containing 3% DMF supplemented with trehalose (400 or 500 mM) or sucrose (300 or 500 mM). Post-thaw sperm quality was assessed using CASA and flow cytometry parameters, together with targeted qPCR analysis of BAK1 and HSP60. Trehalose, particularly at 500 mM, was associated with a more favorable overall functional profile, including higher motility and lower DNA fragmentation. It also showed lower lipid peroxidation- and apoptosis-related values in selected comparisons. However, the response varied depending on the parameter assessed, with S300 showing higher values for mitochondrial membrane potential. BAK1 and HSP60 expression increased after cryopreservation, but no differences were detected between the cryopreserved control and trehalose 500 mM groups, suggesting that these transcriptional changes mainly reflected the freeze-thaw process rather than a trehalose-specific molecular response. Overall, 500 mM trehalose combined with reduced DMF showed a promising functional profile for chicken semen cryopreservation, while the targeted molecular findings provide complementary insight into freeze-thaw-associated stress responses.