Ultra-rapid freezing yields a higher cryoresistance than the conventional slow freezing of epididymal guinea pig (Cavia porcellus) spermatozoa

Ultra-rapid (UR) freezing represents a promising alternative to conventional-slow (CS) freezing of spermatozoa in certain species, with the potential to reduce post-thaw cryodamage. This study evaluated the quality of guinea pig (Cavia porcellus) epididymal spermatozoa cryopreserved by either CS or UR freezing. Twenty sexually mature, clinically healthy males were subjected to bilateral orchiectomy. Epididymal sperm samples recovered by retrograde flushing from the right testes were conventionally frozen by exposing them to liquid nitrogen (LN ₂) vapors, while samples from the left testes were ultra-rapidly frozen by direct immersion of 20 μl droplets into LN ₂. The TCG-EY medium (tris, citric acid, glucose, and 10% egg yolk, 1% BSA) was supplemented with 3% glycerol and 3% dimethylformamide for the CS freezing method, whereas 100 mM sucrose was added for the UR freezing procedure. Both methods drastically reduced the kinematic variables and membrane integrity after cryopreservation (P < 0.01). However, the total and progressive motilities, curvilinear and straight-line velocities with rapid progression, and lateral head displacement were significantly higher in the UR group than in the CS group (P < 0.05). Moreover, sperm viability and acrosomal and DNA integrity were higher after UR freezing than after CS freezing (P < 0.05). In conclusion, ultra-rapid freezing provided better preservation of sperm motility, membrane function, and DNA integrity than conventional slow freezing, indicating greater cryoresistance in guinea pig epididymal spermatozoa. These findings represent a valuable advancement for the cryopreservation of sperm in this species.