The myriad insults that spermatozoa are exposed to during cooling, freezing and thawing result in reduced mitochondrial membrane potential (MMP) and increased apoptotic-like changes. We hypothesise that reduced membrane fluidity during cooling inhibits ion channel regulation, allowing intracellular calcium levels to rise, initiating formation of the mitochondrial permeability transition pore (mPTP). In somatic cells, mPTP formation leads to mitochondrial membrane depolarisation and superoxide leakage, along with the release of pro-apoptotic factors, such as cytochrome c. As these events may be responsible for the symptoms observed in post-thaw spermatozoa, the aim of this project was to investigate the phenomenon of mPTP formation and the effect of mPTP inhibition on stallion spermatozoa. Flow cytometric analysis of spermatozoa pre-loaded with Fluo-3 revealed a significant increase in calcium levels during chilling (2.8 fold from 22 to 2°C over 50 min; P≤0.001). Spermatozoa pre-loaded with Calcein-AM were then chilled over a 24 h period during which a significant loss of fluorescence was observed (to 27.8% of t=0 h; P≤0.001), indicating mPTP formation. Additionally, Western blot analysis revealed a significant loss of cytochrome c following chilling and cryopreservation (36.1±4.5%; P≤0.001 and 46.2±4.0%; P≤0.01 of fresh spermatozoa respectively). As JC-1 staining requires incubation above RT, ionomycin was used as a model to mimic the aforementioned intracellular calcium increase observed during cooling. While ionomycin (10 μM) abolished MMP (0.82% high MMP compared to 89.85% for the control; P≤0.001), pre-treatment with 20 μM cyclosporine A prior to ionomycin exposure reversed this effect such that there was no significant difference to the control (77.56±8.43% vs 89.95±2.44% respectively). As cyclosporine A is a known mPTP inhibitor, this result suggests that mPTP formation does occur in stallion spermatozoa and that cyclosporine A may be a useful additive to incorporate into chilling and cryostorage media to ameliorate the downstream effects of mPTP formation.