Differences in SCSA outcome among boars with different sperm freezability

Abstract

Spermatozoa from some boars sustain the process of cryopreservation poorly and yield poor fertility after artificial insemination. Poor freezability has not been disclosed using conventional semen analyses. A defective chromatin can, if present in a substantial number of spermatozoa, affect the fertilizing ability of spermatozoa. Here we tested the hypothesis that nuclear DNA instability could explain differences in freezability among boars, and complement or supersede conventional tests for sperm quality such as sperm motility or membrane assessments. Frozen-thawed (FT) spermatozoa from a total of 44 stud boars were assessed by the sperm chromatin structure assay (SCSA), in relation to computer-assisted sperm analysis-derived sperm motility variables and sperm viability (triple fluorescent microscopic staining), including three experiments. The first trial, including 24 boars, evaluated the relationship between the sperm motility and viability with levels of DNA integrity. The SCSA showed that most spermatozoa had intact DNA [levels of DNA fragmentation index (%DFI) ranging from 0.63% to 11.85%] significantly correlated (albeit weakly) with current sperm quality variables. The second trial, on 15 boars, assessed the influence of two different thawing rates (20 s at 37 degrees C vs. 8 s at 70 degrees C) and the post-thaw incubation times (0, 30, 150 and 300 min) at 37 degrees C on FT-boar sperm quality. The highest sperm survival (p < 0.05) and the lowest DNA damage (p < 0.01) were achieved when thawing was carried out at 70 degrees C for 8 s, without any change during the first 150 min of incubation. Finally, the third experiment studied if differences in sperm freezability showed by stud boar semen, as 'good' or 'bad' freezers by conventional analyses, could be attributed to differences in chromatin structure. All SCSA parameters were low, but significantly (p < 0.05-0.001) higher for 'bad' freezers, showing they had less homogeneous sperm chromatin than the 'good' freezers. The results indicate that SCSA outcome complements conventional assessment of FT-boar spermatozoa, disclosing differences in their ability to sustain freezing and thawing. However, the low overall DNA damage observed in FT spermatozoa seems to have poor biological significance.