Ferroptosis emerges as the predominant form of regulated cell death in goat sperm cryopreservation

Freezing-induced sperm damage, often associated with oxidative stress, can result in regulated cell death. Given that oxidative stress can trigger various forms of regulated cell death, the prevailing form during sperm cryopreservation remains unknown. Our study aimed to investigate this issue using cashmere goats as a model. We found a significant increase in lyso-phospholipids in frozen-thawed sperm suggested ferroptosis. Assessment of cryopreserved sperm, with or without prior treatment with ferroptosis or apoptosis inhibitors, demonstrated the significant efficacy of ferroptosis inhibitors in reducing freezing damage. This implicates ferroptosis as the primary form of regulated cell death induced during sperm cryopreservation. Additionally, the positive rate of transferrin receptor protein 1 was significantly lower in fresh live sperm (47.8%) than in thawed live sperm (71.5%), and the latter rate was lower than that in dead sperm (82.5%). By contrast, cleaved caspase-3 positivity showed no significant difference between fresh live sperm and thawed live sperm but was notably lower in thawed live sperm than in dead sperm. Our findings establish ferroptosis as the dominant regulated cell death form during goat sperm cryopreservation, providing novel insights into freezing-induced sperm damage mechanisms. These findings have significant implications for optimizing cryopreservation protocols and enhancing sperm viability after freezing-thawing.