Liposome‑based extender consistently improves post-thaw semen quality in rams

Cryopreservation of ram spermatozoa is essential for genetic improvement and germplasm conservation but remains limited by cryo-induced post-thaw deterioration. Conventional egg-yolk-based (EY) extenders suffer from biological variability and biosecurity concerns, motivating the development of chemically defined alternatives. This in vitro study compared a commercial liposome-based extender (LB; OptiXcell®, IMV Technologies) with an EY extender for ram semen cryopreservation using a split-ejaculate design across two independent experiments (42 ejaculates; contrasting management and seasonal conditions). Post-thaw quality was assessed by computer-assisted sperm analysis (CASA; total and progressive motility, VAP, VCL, VSL), flow cytometry (plasma membrane integrity, acrosome integrity, mitochondrial membrane potential), and the hypo-osmotic swelling test (HOST). LB-cryopreserved semen showed higher post-thaw total motility (Experiment I: 34.8 ± 4.2% vs 25.1 ± 3.5%; Experiment II: 40.8 ± 4.1% vs 34.3 ± 4.1%) and progressive motility, with velocity parameters significantly improved. A greater proportion of spermatozoa with intact plasma membranes (Experiment I: 41.9 vs 27.4%; Experiment II: 51.5 vs 37.0%) and preserved acrosomes was detected in LB-treated samples. Mitochondrial membrane potential was higher with LB in Experiment II only (73.5 ± 4.2% vs 59.3 ± 4.2%), reflecting the context-dependent sensitivity of this endpoint. No difference was detected by HOST. Despite inter-individual variability, 94% of ejaculates exhibited higher plasma membrane integrity and two-thirds showed higher total motility with LB. These findings indicate that chemically defined liposome-based extenders improve multiple in vitro indicators of ram sperm function, whilst in vivo fertility trials remain necessary to confirm reproductive relevance.