Waterfowl semen cryopreservation technology is a key link in genetic resource conservation and artificial breeding, but poultry spermatozoa, due to their unique morphology and biochemical properties, are prone to oxidative stress during freezing, resulting in a significant decrease in vitality. In this study, we first used four different freezing procedures (P1–P4) to freeze duck semen and compared their effects on duck sperm quality. Then, the changes in antioxidant indexes in semen were monitored. The results showed that program P4 (initial 7 °C/min slow descent to −35 °C, followed by 60 °C/min rapid descent to −140 °C) was significantly better than the other programs (p < 0.05), and its post-freezing sperm vitality reached 71.41%, and the sperm motility was 51.73%. In the P1 and P3 groups, the sperm vitality was 65.56% and 53.41%, and the sperm motility was 46.99% and 31.76%, respectively. In terms of antioxidant indexes, compared with the fresh semen group (CK), the activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-px) in the P2 group were significantly decreased (p < 0.05), while the activities of SOD and CAT in the P4 group showed no significant changes (p > 0.05) except that the activity of GSH-px was significantly decreased (p < 0.05). And the CAT and GSH-px activities in the P4 group were significantly higher than those in the P2 group (p < 0.05). The content of malondialdehyde (MDA) in the P2 group was significantly higher than that in the fresh semen group (p < 0.05), and there was no significant difference between the P2 group and the P4 group (p > 0.05). The total antioxidant capacity (T-AOC) content of the P2 and P4 groups was significantly lower than that of the fresh semen group (p < 0.05). The staged cooling strategy of P4 was effective in reducing the exposure time to the hypertonic environment by balancing intracellular dehydration and ice crystal inhibition, shortening the reactive oxygen species accumulation and alleviating oxidative stress injury. On the contrary, the multi-stage slow-down strategy of P2 exacerbated mitochondrial dysfunction and the oxidative stress cascade response due to prolonged cryogenic exposure time. The present study confirmed that the freezing procedure directly affects duck sperm quality by modulating the oxidative stress pathway and provides a theoretical basis for the standardization of duck semen cryopreservation technology.

Waterfowl semen cryopreservation technology is a key link in genetic resource conservation and artificial breeding, but poultry spermatozoa, due to their unique morphology and biochemical properties, are prone to oxidative stress during freezing, resulting in a significant decrease in vitality. In this study, we first used four different freezing procedures (P1-P4) to freeze duck semen and compared their effects on duck sperm quality. Then, the changes of antioxidant indexes in semen were monitored. The results showed that program P4 (initial 7°C/min slow descent to -35℃, followed by 60°C/min rapid descent to -140℃) was significantly better than the other programs (P < 0.05), and its post-freezing sperm vitality reached 71.41%, and the sperm motility was 51.73%. In terms of antioxidant indexes, compared with the fresh semen group (CK), the activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-px) in the P2 group were significantly decreased (P < 0.05), while the activities of SOD and CAT in the P4 group showed no significant changes (P > 0.05) except that the activity of GSH-px was significantly decreased (P < 0.05). And the CAT and GSH-px activities in P4 group were significantly higher than those in P2 group (P < 0.05). The content of malondialdehyde (MDA) in P2 group was significantly higher than that in fresh semen group (P < 0.05), and there was no significant difference between P2 group and P4 group (P > 0.05). The total antioxidant capacity (T-AOC) content of P2 and P4 groups was significantly lower than that of fresh semen group (P < 0.05). The staged cooling strategy of P4 was effective in reducing the exposure time to the hypertonic environment by balancing intracellular dehydration and ice crystal inhibition, shortening the reactive oxygen species accumulation and alleviate oxidative stress injury. On the contrary, the multi-stage slow-down strategy of P2 exacerbated mitochondrial dysfunction and oxidative stress cascade response due to prolonged cryogenic exposure time. The present study confirmed that the freezing procedure directly affects duck sperm quality by modulating the oxidative stress pathway, and provides a theoretical basis for the standardization of duck semen cryopreservation technology.