Comparison of cryoprotectant agents and their concentrations on the post-thaw quality of alpaca (Vicugna pacos) epididymal spermatozoa

Cryopreservation of spermatozoa requires the use of cryoprotective agents (CPAs) to minimize membrane damage and preserve cell function after thawing. In South American camelids, glycerol (GL), ethylene glycol (EG), and dimethyl sulfoxide (DMSO) are the most widely used CPAs. However, some studies in other species have explored the potential of amide-based CPAs, such as dimethylacetamide (DMA), dimethylformamide (DMF), and methylformamide (MF). This study aimed to evaluate the interaction between CPA type and concentration on post-thaw sperm quality in alpacas using a 6 × 3 factorial design (six CPAs × three concentrations: 1 %, 3.5 %, and 7 %). Post-thaw assessments included total motility (bright-field microscopy), viability (SYBR14/PI), and mitochondrial membrane potential (MMP) (MitoTracker Deep Red), with the latter two measured by imaging flow cytometry. Data were analyzed using two-way ANOVA to determine main effects and interactions. CPA concentrations of 1 % and 3.5 % produced significantly higher values for motility, viability, and MMP compared to 7 % (P < 0.05). DMSO and GL exhibited significantly higher post-thaw motility (P < 0.05) than DMA and DMF, although no significant differences among CPAs were observed for viability or MMP. In conclusion, CPA concentration has a greater impact than CPA type on post-thaw sperm quality, and concentrations between 1 % and 3.5 % are optimal for preserving motility, viability, and mitochondrial function in alpaca epididymal spermatozoa.