Antifreeze Proteins from Pine Needles Protect Rooster Sperm Cryopreservation via Ultrastructural Stabilization and Thermodynamic Modulation

ABSTRACT Rooster sperm is highly vulnerable to cryodamage, which severely compromises fertility preservation and necessitates the development of efficient cryoprotective agents. In this study, a novel antifreeze protein (AFP) exhibiting thermal hysteresis activity, similar to that of the AFP extracted from pine needles (PN), was chemically synthesized. However, the feasibility and protective mechanisms of PN-AFP as a cryoprotectant for Piao rooster sperm remain incompletely understood. Therefore, this study aimed to systematically investigate its application potential, protective effects, and underlying mechanisms. PN-AFP was supplemented into freezing extenders at concentrations ranging from 0.01 to 0.06 mg/mL, followed by programmed freezing and thawing protocols. Sperm motility, acrosomal and plasma membrane integrity, mitochondrial activity, antioxidant capacity, ultrastructural morphology, fertilization performance, and thermodynamic properties were comprehensively assessed. The results demonstrated that frozen-thawed sperm treated with 0.04 mg/mL PN-AFP exhibited significantly enhanced motility, survival rate, membrane and acrosomal integrity, antioxidant capability, and mitochondrial function compared to all other groups (p < 0.05). Notably, PN-AFP effectively safeguarded sperm ultrastructure from cryoinjury and alleviated surface damage. Cryomicroscopic observations confirmed that PN-AFP efficiently inhibited the formation and growth of large ice crystals within the extenders. Differential scanning calorimetry further verified a marked reduction in crystallization temperature following PN-AFP treatment. Additionally, PN-AFP supplementation substantially improved the fertilization and hatching rates of post-thaw sperm in artificial insemination trials. In conclusion, PN-AFP serves as an excellent novel cryoprotectant for Piao rooster sperm, demonstrating considerable practical value for avian germplasm conservation and the breeding of new crossbred varieties.