In this paper we review the main factors that may affect the evaluation of sperm motility through computer-aided sperm analysis (CASA-Mot) related to the sample, slide, microscope, hardware, software and user. The take-home message of this article is that both commercial and non-commercial CASA systems are available, and all are appropriate for routine analysis. However, in all cases, standard protocols for evaluation and methods for system validation are required.

Computer-aided sperm motility analysis systems have broad scientific and practical relevance. The sources of variation and current applications of these systems and their relationships with other sperm quality tests are discussed. There is still a great need for standardisation in many species, and future systems will probably be able to integrate several sperm quality parameters with motility.

Sperm motility is nowadays considered the best biomarker for fish spermatozoa, becoming a powerful and useful tool for aquaculture and ecological purposes. A high number of sperm kinetic parameters from more than 170 fish species has been reported in more than 700 scientific articles, covering a wide range of topics. This review provides an overview of the major research areas in which fish sperm motility assessment by computer-aided sperm analysis (CASA) has been used successfully.

Fish spermatozoon can reach egg and contribute to fertilization only due to motility phenomenon. This activity is provided by sperm flagellum, playing a double role: a motor and a propeller. A detailed analysis of the flagella performance is a key to the crucial information on qualitative characteristics of sperm and the mechanisms underlying both the spermatozoa movement per se and their targeted approaching towards the egg.

In this article we show how exploiting the rapid (<5 s) stimulatory effect of bicarbonate on boar spermatozoa can be used experimentally to investigate how sperm motility is controlled by molecules of the female reproductive tract. This includes motility inhibition, which can most easily be measured after first using bicarbonate to induce maximal sperm activation. In a wider context, this approach lends itself to the development of sperm motility-based assays for toxicity testing in many different fields.

Despite the existence of computer-aided sperm analysis (CASA) for many years, clinical use among ‘fertility laboratories’ remains limited, despite clear weaknesses in manual semen analysis. Early CASA systems failed to instil confidence and provide a reliable sperm count. However, recent CASA-Mot systems have been validated to provide sperm concentration, motility and velocity, and the benefits related to objectivity and reproducibility should be made clearer.

Modern sperm analysis allows motility or morphometric data to be obtained for individual cells. However, advanced statistical analysis is needed to obtain useful information from these huge datasets. In this review we describe several statistical techniques that can be used for that purpose. We also make several recommendations to promote standardisation of the protocols for acquisition and analysis.

Computer-aided sperm analysis (CASA) has become a powerful tool to quantitatively analyse sperm structure and function of almost any mammalian species. Fully automated analysis was used to describe and assess sperm motility and sperm morphology in primates, domestic animals, laboratory rodents and wildlife. The results provide novel baseline information to be used as standard sperm characteristics and highlight various research and clinical applications.

The microscopic observation at low magnification of a drop of undiluted ram semen shows a collective motion of spermatozoa called wave motion and is used as a semen quality assessment. Objective assessment methods of semen turbulence based on optical flow and fluorescent bead motion showed similar efficiency as subjective wave motion score to predict fertility after insemination. This objective method of semen quality assessment could be used in species such as bovine, sheep, goat and domestic birds.

This study quantified sperm concentration and motility in primarily broadcast spawners in three invertebrate phyla using computer-aided sperm analysis (CASA). CASA technology was used to study sperm motility and quantitatively describe different helical swimming patterns found in the species studied. Baseline information regarding several motility characteristics of invertebrate species is provided, and related to the fertilisation biology in different micromarine environments.

The study of sperm subpopulations may have functional and practical implications. The possible association between kinematic and fluorescent sperm subpopulations was investigated in bulls, observing that the distribution of kinematic sperm subpopulations was different between the fluorescent sperm groups, although the correlation between them was low. There is still a great need for more integrative methods able to simultaneously evaluate different aspects of sperm integrity and functionality.