Computer-Aided Sperm Analysis Protocol for Evaluating Sperm Motility in Japanese Medaka

Changes in sperm motility can serve as an early indicator of reproductive effects caused by environmental chemicals or genetic perturbations. However, sperm motility is highly sensitive to external factors such as osmolarity, ionic composition, and the timing of measurement after activation, making it challenging to obtain consistent and reproducible measurements. Here, we present a standardized protocol for assessing sperm motility in Japanese medaka (Oryzias latipes) using a sperm motility analysis system (SMAS), an application for computer-aided sperm motility analysis (CASA). This protocol details the procedures for sperm collection, activation, and quantitative motility assessment, with particular focus on changes in the percentage of motile sperm post activation and the effects of sperm cryopreservation. We demonstrate time-dependent declines in sperm motility and velocity, and highlight the importance of early post-activation measurements to accurately capture peak motility. Notably, cryopreservation significantly accelerated the decline in sperm motility rate without affecting the initial proportion of motile sperm. To enable reliable comparisons among experimental groups, we recommend standardizing the initiation time after sperm activation by using CASA, and show that measurements should be initiated within 1 min after activation to obtain consistent and reliable data. This standardized SMAS-based protocol provides a robust and reproducible framework for sperm motility analysis in medaka and will be valuable not only for studies in reproductive biology, toxicology, and environmental risk assessment but also for applied research, such as breeding of aquacultural fishes.