In this work, an approach based on digital holography (DH) combined with Raman spectroscopy (RS) is used for a complete label-free analysis of single bovine spermatozoa. DH allows to obtain high-resolution images of bovine sperm from the reconstruction of a single acquired hologram, highlighting in some cases morphological alterations. Quantitative 3D reconstructions of sperm head, both normal and anomalous, have been studied and an unexpected structure of the post-acrosomal region of the head has been detected. Raman imaging analysis have also confirmed such anomalies, suggesting the protein vibrations as associated Raman marker of the defect.