**1. Introduction**

Following the advent of human *in vitro* fertilization [1], much attention has been given to understand both the spermatozoa morphological alterations and the kinematics/dynamics of the swimming spermatozoa [2–11]. In fact, semen analysis is commonly employed both in human and in the zoo-technic field. In the first case, the analysis is mainly applied to study the couple's infertility or to confirm success of male sterilization procedures. Moreover, several studies have shown that for infertile men, the risk for developing a testicular cancer is slightly higher-than-average. So, independently of the will to have children, male fertility is a good

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

indicator for general health. On the other hand, in the zoo-technic field, animal semen analysis is commonly used in animal production laboratories and reproductive toxicology.

The main requirements for the development of techniques used for an accurate semen analysis are the following:


The sperm cell is almost transparent in conventional bright field microscopy, as its optical proprieties differ slightly from the surrounding liquid, generating little contrast. On the other hand, a light beam that passes through a spermatozoon undergoes a phase change, in comparison with the surrounding medium, the amplitude of which depends on the light source, the thickness, and the integral refractive index of the object itself. A qualitative visualization of this phase contrast may be obtained by contrast interference microscopy (phase contrast or Nomarski/Zernicke interferential contrast microscopy). However, it is difficult and timeconsuming to obtain a quantitative morphological imaging. In fact, a fine z-movement of the biological sample is required in order to acquire a collection of different planes in focus. This collection of acquired images is used in postelaboration to produce a 3D image of the object under investigation [12]. The same approach has been used to obtain information about sperm motility. Nevertheless, this 2D intrinsic analysis implies a partial in-plane representation of the motility features due to difficulty to track the 3D spatial motion of spermatozoa that quickly move out of focus. In order to overcome these intrinsic limitations, several approaches have been recently developed. In this contest, the optical approaches are deeply investigated.

In particular, over the last few years, holographic imaging in microscopy has been established as a valid noninvasive, quantitative, label-free, high-resolution, and phase-contrast imaging technique. So this chapter tries to summarize the state-of-art on the semen analysis and recent achievements obtained by a holographic imaging [13–17]. We will show that the unique potentialities of the holographic imaging have been used to provide structural information on both the morphology and the motility of sperm cells [18]. Moreover, the combination of the holographic technique with others approaches, such as the Raman spectroscopy, will be described, too. In fact, spermatozoa from infertile men could present a variety of alterations (such as alterations of chromatin organization [19], aneuploidy [20], and DNA fragmentation [21]) that can decrease reproductive capacity of men. Current methods of DNA assessment are mainly based on fluorescence microscopy, and thus samples are unusable after the analysis [22–25]. Therefore, the ability to simultaneously analyze, in a nondestructive and noninvasive way, both the morphology and biochemical functionalities of the spermatozoa could bring greater understandings [26]. Thus, the chapter will allow a bird's-eye view into the potentiality of the semen analysis performed by means of the holographic imaging, showing that this approach is extremely important for the intracytoplasmic sperm injection (ICSI) procedure, where it is highly required the development of a method that allows characterizing and directly select the best spermatozoon to inject into the oocytes [9, 27].
