Application of flow cytometry using advanced chromatin analyses for assessing changes in the sperm structure and DNA integrity in a porcine model

Chromatin status is critical for sperm fertility. We tested a multivariate approach for studying pig sperm chromatin, aiming to capture the chromatin structure's complexity with a set of quick and simple techniques, not only DNA damage. Sperm doses from 36 boars (3 ejaculates/boar) were analyzed at days 0 and 11 (cooled storage). Analyses were: CASA (motility) and flow cytometry to assess sperm functionality and chromatin structure by SCSA (DNA fragmentation %DFI and chromatin maturity %HDS), monobromobimane (mBBr, tiol status/disulfide bridges between protamines), chromomycin A3 (CMA3, protamination) and 8-hydroxy-2'-deoxyguanosine (8-oxo-dG, DNA oxidative damage). Data were analyzed by linear models for effects of boar and storage, correlations, and multivariate analysis as hierarchical clustering and principal component analysis (PCA). Storage reduced sperm quality parameters, mainly motility, with no critical oxidative stress increases, while chromatin status worsened slightly (%DFI and 8-oxo-dG  increased while mBBr MFI and disulfide bridges decreased). Boar significantly affected most chromatin variables except for CMA3, with storage affecting most except %HDS. At day 0, sperm chromatin variables clustered closely, except for CMA3, and %HDS and 8-oxo-dG correlated with many variables (notably, mBBr). After storage, the relation between %HDS and 8-oxo-dG remained, but correlations among other techniques disappeared, and mBBr variables clustered separately. The PCA suggested a considerable influence of mBBr on sample variance, especially regarding storage, with SCSA and 8-oxo-dG affecting between-sample variability. Overall, CMA3 was the least informative, in contrast with results in other species. The combination of DNA fragmentation, DNA oxidation, chromatin compaction, and tiol status seems a good candidate for obtaining a complete picture of the pig sperm nucleus status, raising many questions for future molecular studies and deserving further research to establish its usefulness as fertility predictors in multivariate models. The meaning of CMA3 should be clarified.