5-aminolevulinic acid improves drake post-thaw sperm quality via enhancing mitochondrial function

Cryopreservation of sperm is an essential approach for the preservation and utilization of poultry genetic resources. However, during the freezing process, drake sperm often suffers structural damage caused by low-temperature stress and physical injury, which impairs mitochondrial function and markedly reduces post-thaw sperm quality and fertilizing ability. In this present study, different concentrations of 5-aminolevulinic acid (5-ALA; 0, 100, 250, 500, and 1000 μM) were supplemented into the freezing extender, and several key parameters of post-thaw drake sperm were evaluated to investigate the potential role of 5-ALA in semen cryopreservation. The results demonstrated that supplementation with 500 μM 5-ALA significantly improved post-thaw sperm motility, acrosome integrity, and viability compared to the control group (P < 0.05). Further analyses revealed that treatment with 500 μM 5-ALA significantly increased the heme levels in sperm (P < 0.05), enhanced the activity of mitochondrial complex IV, and elevated both mitochondrial membrane potential (MMP) and adenosine triphosphate ATP levels (P < 0.05), indicating that 5-ALA facilitated mitochondrial energy metabolism. In addition, 500 μM 5-ALA treatment significantly reduced sperm reactive oxygen species (ROS) levels, lipid peroxidation (LPO) content, apoptosis levels and DNA fragmentation (P < 0.05). This indicates that 5-ALA can reduce the damage caused by oxidative stress during freezing, inhibit LPO reactions, indirectly protect the integrity of sperm membrane and nucleus, and thus maintain the survival ability of sperm. Taken together, these findings indicate that supplementation with an appropriate concentration of 5-ALA effectively supports the maintenance and recovery of mitochondrial function. Thereby improving the efficiency of the electron transport chain and enhancing ATP production to meet the energy demands of sperm. Moreover, by alleviating the damage caused by oxidative stress to sperm, the integrity of sperm structure has been stabilized, and ultimately contributes to the enhancement of post-thaw sperm motility.