A new method to analyze boar sperm DNA fragmentation under bright-field or fluorescence microscopy

Abstract

We present a new, rapid and simple method to study DNA Fragmentation Index (DFI) in sperm samples from boar under bright-field and fluorescence microscopy. Discrimination of sperm cells containing fragmented DNA relies on the extreme peripheral diffusion of their chromatin fragments, whereas those sperm nuclei without DNA fragmentation do not disperse or show very restricted spreading of DNA loops close to the flagellum. The basic methodology provided in the commercial kit Sperm- Sus-Halomax ® allows, in addition to a direct estimation of DFI in a sperm sample under bright field microscopy, a direct visualization of DNA breaks by incorporation of labelled nucleotides using the DNA polymerase I following the in situ nick translation assay (ISNT methodology not provided in the kit). An external control using DBD-FISH (DNA breakage detection-fluorescence in situ hybridization) on human and boar sperm samples was used in this experiment. The results obtained show (i) low levels of background DNA fragmentation (from 0.7 to 10%), (ii) no significant differences for DFI after the application of Sperm- Sus-Halomax and ISNT, with a tendency to be underestimated after using DBD-FISH and (iii) a characteristic chromatin organization in boar sperm nucleus, with a particular response to chromatin loop relaxation and preferential DNA labelling by ISNT at the proximal nuclear area, close to the flagellum. This methodology allows the routine assessment of boar sperm samples for DFI, as well as basic and clinical research on this relevant topic in any laboratory of semen analysis.