Evaluation of dimethylformamide (DMF) and Trehalose as cryoprotectants in African penguin Spheniscus demersus semen cryopreservation

We investigated the use of dimethylformamide (DMF), a permeating cryoprotectant, and its combination with trehalose, a non-permeating cryoprotectant, for the cryopreservation of African penguin (Spheniscus demersus) semen. Semen samples were cryopreserved using either 6% DMF alone (control group) or 3% DMF with 500 mM trehalose (T500 group). Post-thaw sperm quality was assessed based on motility parameters, viability, mitochondrial membrane potential, acrosomal integrity, apoptosis, and DNA fragmentation. Each treatment exhibited significant differences in specific aspects of post-thaw sperm quality. The control group showed higher total motility and mitochondrial membrane potential, whereas the T500 group demonstrated improved kinematic parameters (VCL, VSL, VAP, ALH, BCF), reduced apoptotic-like changes, less acrosomal damage and lower DNA fragmentation. Both treatments maintained viability, with a strong correlation between morphological and fluorescent viability assays. While the addition of trehalose did not enhance total motility, it conferred targeted cellular protection and improved motion dynamics potentially relevant to fertilization. These findings indicate that DMF and trehalose can improve post-thaw semen quality in African penguins, and that cryoprotectants may influence specific sperm traits differently across and within species.