Slow Freezing of Preserved Boar Sperm: Comparison of Conventional and Automated Techniques on Post-Thaw Functional Quality by a New Combination of Sperm Function Tests

SIMPLE SUMMARY: The slow freezing of boar spermatozoa is carried out by exposure to liquid nitrogen vapors (conventional) or by using automated equipment that controls the temperature drop curve. In both cases, freezing/thawing causes a reduction in sperm functionality. Therefore, the objective of this study was to compare both freezing techniques through a panel of evaluations of sperm function by CASA and flow cytometry technologies. We observed the negative effect of post-thawing incubation time on sperm motility. In addition, there was a significant decrease in the levels of free radicals, reduced lipid peroxidation, and a loss of cholesterol and markers of apoptosis, which have an impact on the better conservation of the motility of frozen boar spermatozoa with automated equipment. For this reason, the control of the temperature drop curve allows sperm functionality to be preserved. No significant differences were observed for some parameters associated with the state and preservation of the membranes. ABSTRACT: The slow freezing of boar sperm is the only way to preserve genetic material for extended periods; this can be achieved with exposure to liquid nitrogen vapors (conventional) or by using automated freezing equipment. The aim was to compare the effect of both techniques on post-thaw functionality. Boar sperm devoid of seminal plasma and resuspended in lactose-egg yolk-glycerol medium were cryopreserved. Conventional: straws were exposed to LN(2) vapors; automated: using a drop curve of −39.82 °C·min(−1) for 113 s from −5 to −80 °C during the critical period; and subsequent immersion in NL(2). Cell viability, cholesterol flow, mitochondrial membrane potential (MMP), lipid peroxidation, peroxynitrite, superoxide anion levels, phosphatidylserine translocation, and caspase activation were evaluated by flow cytometry. In addition, total motility (TM) and progressive motility (PM) were determined by the SCA system immediately (T0), 60 (T60), and 120 min (T120) post-thawing. Automated freezing significantly reduces cholesterol flow and free radical and lipid peroxidation levels, making it possible to preserve motility for 120 min of incubation. At the same time, viability, acrosome integrity, MMP, and caspase activation did not differ from the conventional technique. In conclusion, controlling the temperature drop curve using automated freezing equipment reduces oxidative/nitrosative stress, preserving membrane fluidity and sperm motility.