Dog sperm DNA: Raw semen evaluation with Toluidine blue stain

The toluidine blue (TB) stain has been used in different species to evaluate the degree of chromatin condensation. The objectives of this study were as follows: simplify the TB stain to evaluate sperm in canine raw semen, verify the staining patterns for this species using this simplified technique and establish a protocol for using dithiothreitol (DTT) as a positive control for TB staining in dogs. Twenty-one ejaculates were collected from 7 adult male dogs; semen was extended, fixed with ethanol 96° and stained with TB using 2 staining times: 15 and 30 min. In addition, 3 incubation times with 1% DTT were assayed (2, 5 and 30 min). Three staining patterns were established: light blue colouring (TB negative, normal chromatin condensation), light violet (TB intermediate, some degree of chromatin decondensation) and dark blue-violet (TB positive, high degree of chromatin decondensation). No significant differences (p > 0.05) were observed between the staining times (15 and 30 min) for any of the TB patterns. All DTT incubation times (2, 5 and 30 min) showed 100% sperm positive to TB. To conclude, it was possible to simplify the TB stain and determine the different patterns in canine spermatozoa. Also, DTT can be used both as a positive control for the stain and to evaluate individual susceptibility to decondensation in vitro.