Numerical analysis is implemented to investigate biological sample starting from Digital Holographic (DH) recording. The aim is to improve visualization and detection of cow spermatozoa. Digital holograms are recorded in the off-axis geometry where optical setup is a Mach-Zehnder interferometer. Then holograms are numerically manipulated to retrieve, besides the usual Quantitative Phase Map (QPM), Differential Interference Contrast (DIC) visualization. Furthermore, a new approach, named digital self-referencing holography, is described it’s able to accomplish quantitative phase analysis especially useful for specimen flowing in microfluidic channels.