Dose-dependent effects of Nymphaea lotus extract in semen extenders on sperm quality and fertility in Thai native roosters during cooled and cryopreserved storage

This study evaluated the dose-dependent effects of Nymphaea lotus extract supplementation in semen extenders on sperm quality and fertility in Thai native roosters under cooled and cryopreserved storage conditions. Phytochemical analysis revealed that the ethanolic extract contained high levels of total phenolics (596 mg/g DW) and proanthocyanidins (295 mg/g DW), along with flavonoids and tannins. In vitro antioxidant assays demonstrated strong radical-scavenging and ferric-reducing activities, with IC₅₀ values comparable to standard antioxidants, and significant protection against AAPH-induced hemolysis. For cooled semen stored at 5°C, supplementation with 0.10 mg/mL N. lotus significantly improved sperm motility and viability and reduced malondialdehyde levels for up to 48 h (P < 0.05), with fertility significantly enhanced at 24 h of storage. Although fertility at 48 h did not differ significantly from the control group, absolute fertility values remained high. In cryopreserved semen, supplementation with 0.025 mg/mL resulted in the highest post-thaw sperm motility, viability, and fertility (P < 0.05). These findings demonstrate that N. lotus extract is an effective natural antioxidant for improving rooster semen quality and fertilizing efficiency, with optimal supplementation levels dependent on the storage method.