Factors influencing post-thaw quality of stallion spermatozoa: Reassessing the role of penetrating cryoprotectants (CPAs)

This study examined interactions between cryoprotectant (CPA) composition and processing factors (i.e., pre-freeze cooling rate, egg yolk concentration, cryopreservation step, final sperm concentration) that may influence post-thaw quality of stallion sperm. In Experiment 1, 24 ejaculates were cryopreserved in extenders with either 20% egg yolk (EY) or 2% egg yolk with added milk proteins (SMEY) and containing either glycerol (Gly) and methyl formamide (MF; Gly + MF), methyl formamide (MF), or dimethylformamide (DF). Post-thaw sperm quality was assessed via computer-assisted motion analysis and flow cytometry. An interaction between egg yolk concentration (EY vs. SMEY) and CPA (Gly+MF vs. MF vs. DF) was observed, whereas an effect of pre-freeze cooling rate (–0.1 vs. –1 °C/min) was not detected. Sperm cryopreserved in EY-Gly+MF exhibited the highest sperm motility parameters and plasma membrane integrity values, as well as the lowest levels of apoptotic and oxidative stress-associated changes. In Experiment 2 (n = 24 ejaculates), sperm quality was not affected immediately after CPA addition (Gly vs. MF vs. Gly+MF) or pre-freeze cooling but decreased significantly after freezing and thawing. Post-thaw motility and viability were similar between Gly and Gly+MF, both higher than for MF. Exposure to hyperosmotic (CPA-containing) or isosmotic (CPA-free) media during analysis at every step of the cryopreservation process had no impact on sperm quality. In Experiment 3 (n = 24 ejaculates), sperm cryopreserved at 200, 400, or 800 ×10 6 sperm/mL exhibited higher post-thaw motility and viability than sperm cryopreserved at 50 or 100 ×10 6 sperm/mL, regardless of CPA.